Seminar

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ThalassaemiaAli T Taher, David J Weatherall, Maria Domenica Cappellini

Inherited haemoglobin disorders, including thalassaemia and sickle-cell disease, are the most common monogenic diseases worldwide. Several clinical forms of α-thalassaemia and β-thalassaemia, including the co-inheritance of β-thalassaemia with haemoglobin E resulting in haemoglobin E/β-thalassaemia, have been described. The disease hallmarks include imbalance in the α/β-globin chain ratio, ineffective erythropoiesis, chronic haemolytic anaemia, compensatory haemopoietic expansion, hypercoagulability, and increased intestinal iron absorption. The complications of iron overload, arising from transfusions that represent the basis of disease management in most patients with severe thalassaemia, might further complicate the clinical phenotype. These pathophysiological mechanisms lead to an array of clinical manifestations involving numerous organ systems. Conventional management primarily relies on transfusion and iron-chelation therapy, as well as splenectomy in specific cases. An increased understanding of the molecular and pathogenic factors that govern the disease process have suggested routes for the development of new therapeutic approaches that address the underlying chain imbalance, ineffective erythropoiesis, and iron dysregulation, with several agents being evaluated in preclinical models and clinical trials.

IntroductionSince the first published description of severe thalass­aemia over 90 years ago by Cooley and Lee,1 several accounts of the disease have been described and an extensive amount has been learnt.2–4 Although the cellular and molecular basis of this group of diseases was initially unknown, in the past 50 years a considerable amount has been discovered to create a substantial body of work.5 Using these resources we now have a refined under­standing of the pathophysiology of the thalassaemia syndromes. However, despite our understanding of the pathophysiology, management of these diseases has been complex and is progressing gradually.3,4 Additionally, the available therapeutic routes for thalassaemias and the complications that result from current treatments are few. Although a great deal of excitement has developed around newer therapeutic approaches and potential curative strategies,6 much remains to be understood about the clinical variability of these disorders, the natural history of the thalassaemia syndromes, and the optimal use of the currently available treatments. In this Seminar, we aim to provide an overview of the thalass aemia syndromes and comprehensively discuss our current approach to the clinical management of these diseases. As will become evident, this under standing is crucial to ensuring that new therapies can be effectively integrated into the repertoire of existing management strategies.

Thalassaemia, sickle­cell disease, and other inherited haemoglobin disorders are the most pervasive mono ­genic diseases worldwide. The high frequency of in­herited haemoglobin variants in certain regions reflects their heterozygote resistance to Plasmodium falciparum malaria, and extensive studies7 have shown that this resis tance is certainly the case for α­thalassaemia, β­ thalassaemia, and haemoglobin E. An estimated 1–5% of the global population are carriers for a genetic thalassaemia mutation.8–14 Although the epidemiology of the various clinical forms remains poorly recognised, the disease is known to be highly prevalent in the area extending from

sub­Saharan Africa, through the Mediterranean region and Middle East, to the Indian subcontinent and east and southeast Asia.8–14 Thus, over 90% of patients with these disorders live in low­income and middle­income countries. The number of patients with these diseases is expected to increase in the coming years as infant mortality from infectious and nutritional causes declines in many regions of the world. As a result, when discussing management strategies, we will often focus on the current best practices, but it is important for clinicians to be aware of the substantial limitations in implementing even these stan­dard therapies to many patients around the world. Therefore we will focus our discussion on the need for improved therapies, as well as on more standardised and easy­to­implement strategies for use in resource­poor countries. It should also be noted that because of continued migration, these diseases are now becoming increasingly common in large multiethnic cities in Europe and North America, making it a global health concern.15–18

Molecular and clinical formsAt the molecular level, haemoglobin synthesis is controlled by two multigene clusters on chromosome 16 (encoding

Published Online July 31, 2017 http://dx.doi.org/10.1016/S0140-6736(17)31822-6

Department of Internal Medicine, American University of Beirut Medical Centre, Beirut, Lebanon (Prof A T Taher MD); MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK (Sir D J Weatherall MD); and Department of Clinical Sciences and Community, University of Milan, IRCCS Ca’Granda Foundation Maggiore Policlinico Hospital, Milan, Italy (Prof M D Cappellini MD)

Correspondence to: Prof Ali T Taher, Department of Internal Medicine, American University of Beirut Medical Center, PO Box 11–0236, Beirut 11072020, Lebanon ataher@aub.edu.lb

Search strategy and selection criteria

We searched PubMed using the terms “thalassaemia OR thalassemia” in combination with “molecular” or “epidemiology” or “diagnosis” or “pathophysiology” or “clinical complications” or “treatment OR management”. We limited our search to publications in English. We mostly selected publications from January, 2006, to May, 2017, but did not exclude frequently referenced and highly regarded older publications. We also searched the reference lists of articles identified by this search strategy and selected the most relevant ones. Review articles and book chapters are cited to provide readers with more details and more references than can be addressed in this Seminar.

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the α­like globins) and on chromosome 11 (encoding the β­like globins; figure 1).19,20 The genes in such clusters are arranged along the chromosome in the order that they are expressed during development to produce different haemoglobin tetramers during embryonic, fetal, and adult life. The high homology of the α­globin genes can result in unequal­meiotic cross over, which is the basis for the deletions that cause the α­thalassaemias discussed later in this section. Within the β­globin gene cluster, the ε­gene is expressed only in early embryos, and downstream from this gene are two γ­genes, the product of which is found in fetal haemoglobin (Hb F, α2γ2)—the haemoglobin form that predominates throughout most of gestation. The δ­gene product forms a minor haemoglobin component, Hb A2 (α2δ2), which is useful in the diagnosis of the thalassaemias. The β­gene product combines with

α­globin to form Hb A (α2β2), the major haemoglobin component of adult red blood cells. During the first month of gestation, embryonic haemoglobins β2ε2, α2ε2, and β2γ2 are formed in erythroid cells located primarily in the yolk sac. During the remainder of fetal life, the sites of erythropoiesis gradually shift from the liver and spleen to the bone marrow, with red blood cells mainly containing Hb F (α2γ2). A switch from γ­globin to β­globin expression begins before birth and completes by the time the baby reaches 6 months of age. After this time over 95% of the haemoglobin in normal red blood cells is adult Hb A (α2β2), with the remaining haemoglobin consisting of two minor components, Hb A2 and Hb F.19,20 Hb F is usually confined to a small number of red blood cells known as F cells. Underlying molecular defects in the α­globin or β­globin gene clusters form the basis of defective haemoglobin synthesis and the various inherited forms of α­thalassaemias or β­thalassaemias. The type and severity of these clinical forms can also rely on additional and independent intrinsic and extrinsic factors, which will be explained in this section. The thalassaemias can also manifest from co­inheritance of β­thalassaemia and structural haemoglobin variants like haemoglobin S, C, and E.11,21 Several clinically recognisable forms of haemoglobin S/β­thalassaemia and haemoglobin C/β­thalassaemia exist, each of which have unique character istics and management peculiarities;20 however, these will not be covered in this seminar.

β-ThalassaemiaPatients with β­thalassaemia have been typically cate­gorised as minor, major, or intermedia on the basis of their α­globin or β­globin chain imbalance, severity of anaemia, and clinical picture at presentation. Over 200 mutations in the β­globin gene that cause disease have been recognised, ranging from silent mutations (silent β), to mild mutations that cause a relative reduction in β­globin chain production (β+), to severe mutations that result in complete absence of β­globin chain synthesis (β0), with deletions of the gene being uncommon.22 β­Thalassaemia minor (trait or carrier) represents the heterozygous inheritance of a β­thalassaemia mutation, with patients often having clinically­asymptomatic­micro­cytic anaemia, although others can have no identified haematological abnormalities—so­called silent carriers. Patients with β­thalassaemia major usually present with severe anaemia in infancy and become transfusion dependent for life, whereas patients with β­thalassaemia intermedia can present later in life with mild­to­moderate anaemia and variable transfusion requirements.23–25 Both β­thalassaemia major and intermedia can result from the homozygous or compound­heterozygous inheritance of mutations in the β­globin gene.

Several modifications can result in patients having β­thalassaemia intermedia rather than major, including the extent of the α­globin to β­globin chain imbalance, ineffectiveness of erythropoiesis, and the resulting

Figure 1: Globin synthesis at the molecular level(A) Organisation of the α-globin family on chromosome 16 and the β-globin family on chromosome 11. (B) The sites of erythropoiesis and the pattern of globin synthesis during development. ψ designates non-expressing pseudogenes. The three exons of the globin genes are shown in pale blue. Reproduced from Bunn and colleagues19 with permission from McGraw-Hill Education. IVS=intervening segments (or introns).

50B

A

40

30

20

10

Site of erythropoiesis

Bone marrowLiver

Yolk sac

Spleen

Birth

α

γ

β

ε

ξ

β

γ

6 12 302418 636 121 18 24 30 36 4842

Postconceptual age (weeks) Postnatal age (weeks)

Tota

l glo

bin

synt

hesis

(%)

5’

5’ 3’

5 kb

α1 α1α2ψξξ

5’

11

16

3’Gγε ψβ

ψ

1ψβ2 βδΑγ

3’

5’ IVS-I IVS-II 3’

IVS-I IVS-II

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anaemia. The primary modifier is the severity of the mutation or mutations in the β­globin gene. Secondary modifiers include co­inheritance of α­thalas saemia, and increased synthesis of γ­chains and fetal haemoglobin production after infancy.22–26 Some rare forms of β­ thalassaemia intermedia result from deletions removing the upstream­regulatory elements, but leaving the globin genes intact. Other rare mutations linked on the same chromosome have been important in developing our understanding of how the switch from γ­globin to β­globin gene expression is regulated. All these mutations result in variable concentrations of increased γ­globin expression and increased concentrations of fetal haemoglobin, leading to what is known as hereditary persistence of fetal haemoglobin. Other forms of hereditary persistence of fetal haemoglobin result from deletions removing the adult δ­globin and β­globin genes, but leaving at least one γ­globin gene intact (δβ­thalassaemia).19,20 Tertiary modifiers include genetic and environmental factors that alter specific complication rates.23–25,27 β­Thalassaemia intermedia might also result from the increased production of α­globin chains by a triplicated or quadruplicated α­genotype associated with β­heterozygosity, or when a single β­globin locus is affected and the other is completely normal (dominant inclusion body β­thalassaemia).23–25 When β­thalassaemia is co­inherited with the structural variant haemoglobin E (haemoglobin E/β­thalassaemia), which results in β­globin chain synthesis similar to a mild β­gene mutation, the resulting clinical forms can also be of varying severity depending on age and the degree of anaemia at presentation. The clinical forms of haemoglobin E/β­thalassaemia are commonly classified as mild, moderate, and severe, with the severe form being similar to β­thalassaemia major, and the mild and moderate forms being similar to β­thalassaemia intermedia.28,29 The same primary, secondary, and tertiary genetic and environmental modifiers are also relevant in the case of haemoglobin E/β­thalassaemia, and influence the clinical severity of the disease.30,31

α-Thalassaemiaα­Thalassaemia has two main forms, α+­thalassaemia and α⁰­thalassaemia, and their classifications depend on whether one or both of the linked α­globin genes are deleted or reduced in activity by mutation. The two common forms of α+­thalassaemia are designated –α³·⁷ and –α⁴·² to describe the lengths of the underlying deletions. α+­Thalassaemia has several forms that result from point mutations, the most common being caused by the chain­termination mutant haemoglobin Constant Spring, designated αCSα. In their heterozygous state these conditions are silent and in their homozygous state they are characterised by mild­hypochromic anaemia. α⁰­Thalassaemias are usually caused by the deletion of both the linked α­globin genes and are designated by the place of discovery of the first case (eg, –Med and –SEA, reflecting the first cases to have been found in the

Mediterranean region and southeast Asia). The com­pound heterozygous states for α+­thalassaemia and α⁰­thalassaemia, –α/— or αCSα/—, result in a large excess of β­chain production with the formation of β4 tetramers, designated as haemoglobin H. These β4 tetramers are a highly unstable variant of the β­chain and they precipitate in red blood cells causing haemoglobin H disease, which is characterised by variably severe haemolysis and consequent anaemia. The homozygous state for α⁰­thalassaemia, —/—, results in the production of tetramers of γ­chains (γ4) known as haemoglobin Bart’s, named after the hospital in London, UK, where it was first discovered. This homozygous state of α⁰­thalassaemia is associated with a condition called haemoglobin Bart’s Hydrops fetalis, which is usually characterised by death in utero or just after birth. Rarer causes of α­thalassaemias include deletions or mutations of regulatory mutations involving the α­globin gene cluster.4,17

There is another group of α­thalassaemias that, unlike those described above, occur in no particular ethnic groups. They are described under the general title of α­thalassaemia and mental retardation. One thalassaemia in this group is known as ATR­16 syndrome. This syndrome involves the whole α­globin gene complex on chromosome 16 and is associated with mild­to­moderate mental retardation. Another member of this group is known as α­thalassaemia X­linked intellectual disability (ATRX) syndrome and results from mutations of the ATRX gene leading to severe developmental abnormalities, facial dysmorphism, and other disorders in addition to its α­thalassaemia phenotype. Finally, there is another form of mild hemoglobin H disease that occurs predominantly in men and is associated with a form of myelodysplastic syndrome and pre­leukaemia, which has also been found to be associated with mutations involving ATRX.17

Screening and diagnosisScreening and prevention programmes for thalassaemia are now widespread, with their adoption being dependent on regional distribution and cultural factors. Programmes of nationwide screening, and premarital and neonatal screenings have been implemented. Confirmation of a thalassaemia diagnosis requires analysis of red blood cells combined with haemoglobin electrophoresis, with DNA analysis required to confirm the diagnosis of α­thalassaemia and haemoglobin E. Specific guidance on the pathway to establish a thalassaemia diagnosis has been reviewed elsewhere and is available from recent clinical management guidelines.23,32,33

Clinical classificationOver the past decade, a gradual transition in the labelling of the thalassaemias has occurred, moving away from the molecular forms to a more simplified categorisation largely based on clinical­management criteria. Transfusion therapy remains the basis of management for these disorders, and the frequency and magnitude of transfusion

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requirements indirectly reflects the underlying severity of the disease. Moreover, transfusion therapy is not only able to control most of the underlying pathophysiological mechanisms but it also contributes a great deal to secondary morbidity.23,33 Thus, patients today are com­monly categorised as having transfusion­dependent thalassaemia (TDT)—patients who are not capable of producing sufficient haemoglobin to survive without blood transfusions—or non­transfusion­dependent thalassaemia (NTDT).23,33 However, several points of caution should be highlighted when using such cate­gorisations. For instance, patients with NTDT can still require transfusion therapy sporadically, or even regularly, but not for their entire lifetime. This treatment would be for the prevention or management of certain disease manifestations—and will be illustrated in subsequent sections—whereas patients with TDT require lifelong transfusion treatment for survival. Patients with β­thalassaemia intermedia, haemoglobin H disease, and mild­to­moderate forms of haemoglobin E/β­thalassaemia often fall under the classification of NTDT; whereas patients with β­thalassaemia major and severe forms of haemoglobin E/β­thalassaemia are classified as having TDT.23,24,33 Additionally, it should be noted that some patients categorised as having TDT might have been misclassified. For instance, if transfusions had been started to treat low haemoglobin concentrations as a result of an acute complication (eg, an infection) in childhood, and the patient’s transfusion requirements had not been revisited and the transfusions thus never halted.34 Also, it should be highlighted that TDT and NTDT are fluid categories based on clinical variables and a patient might move from one group to another as a result of variations and advances in clinical management, or because of changes in other disease modifiers—thus these designations should primarily represent a patient’s current clinical status with the understanding that the categories are interchangeable.23,24,33,34

Pathophysiology and associated morbidityThe underlying disease process in patients with thalass­aemia remains similar for those patients categorised as

having NTDT or TDT, and the role of transfusion therapy in ameliorating much of these pathogenic mechanisms (while introducing other secondary complications) is best illustrated in studies showing the different morbidity profile between the two catagories.24,35 The hallmarks of the disease—with or without treatment—are the α­globin or β­globin chain imbalance leading to ineffective erythro­poiesis, an array of subsequent pathophysiological mecha­nisms, and a multimorbidity profile (figure 2).24,36 In β­thalassaemia, accumulation of unstable α­globin chain tetramers in erythroid cells leads to premature cell death inside (ineffective erythropoiesis) and outside (peripheral haemolysis) the bone marrow, facilitated by reactive­oxygen­species formation and structural­membrane de­formities leading to senescence­antigen exposure.37,38 The immediate resulting manifestation is chronic­haemolytic anaemia that can result in acute compli cations such as cholelithiasis and short­term and long­term detrimental effects on growth, organ, and vascular function.39 In­creased proliferation of erythroid precursors in the bone marrow leads to medullary expansion and subsequent bone deformities and low bone mass.37 Compensatory haemo poietic points out side the bone marrow are also activated, primarily in the spleen (splenomegaly) and liver (hepatomegaly), but can also be activated in any body tissues with haemopoietic potential, leading to the formation of extramedullary pseudotumours in high­risk anatomic locations such as the spinal canal.40 The ex­posure of erythroid cells to senescence antigens, such as phosphatidylserine, during ineffective erythro poiesis gives them prothrombotic potential. Alongside other abnor malities in platelets and the coagulation system, hyper coagulability and associated vascular mani festations like venous thrombosis and pulmonary hypertension are commonly seen in patients with thalassaemia, especially in patients who have been splenectomised and in whom such markers are further elevated.41 Finally, ineffective erythropoiesis results in increased iron absorption and primary iron overload mediated by the hepatic hormone hepcidin. An erythroid factor has been postulated as communicating to the liver the need of iron for the incoming red blood cells. This factor would be produced

Figure 2: Pathophysiology of patients with thalassaemia syndromes

Marrow expansion and bone disease

Extramedullary haemopoiesis and organomegaly

Peripheral haemolysis and gall stones

Organ damage (heart, liver, endocrine)

Hypercoagulability and vascular disease

α/β-Chain imbalance Ineffective erythropoiesis

Iron overload

Anaemia

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by erythroid cells, especially under the condition of ineffective erythro poiesis, and acts to suppress hepcidin synthesis in the liver, leading to increased intestinal iron absorption and increased release of recycled iron from the reticulo endothelial system. Various erythroid factors have been investigated, including growth differentiation factor 15 (GDF15), twisted­gastrulation 1 (TWSG1), and erythro­ferrone (ERFE); although only ERFE was shown to be increased in animals with β­thalassaemia compared with those without β­thalassaemia.42,43 Such iron overload concen trations can reach clinically significant thresholds and lead to morbidities in various organ systems, especially the liver.44 Notably, both anaemia and iron overload can also further worsen ineffective erythropoiesis and complicate patho physiology.42,43 In patients with α­thalassaemia, the concepts of α­chain to β­chain imbalance and ineffective erythropoiesis is similar to β­thalassaemia. β­Globin chains can form soluble tetramers—β4, or haemoglobin H—that precipitate within the cell forming insoluble inclusions (Heinz bodies) that damage the red blood cell’s membrane. Although downstream manifestations like primary iron overload and hypercoagulability exist, they tend to be milder in patients with α­thalassaemia than in patients with β­thalassaemia, and the key pathological mechanism is peripheral haemolysis and chronic anaemia, rather than ineffective erythropoiesis, as is seen in patients with β­thalassaemia.24,36

Since adequate and regular transfusion therapy can be considered part of the disease profile of patients with TDT, it is fair to say that the majority of these patho ­genic mechanisms and associated morbidities are more commonly encountered in patients with NTDT.24,35 Whereas adverse transfusion­related complications, in­cluding secondary­iron overload and subsequent organ dysfunction, have become hallmarks of clinical mor­bidity in well transfused patients with TDT.45 Clinical aspects of all such morbidities in patients with thalass­aemia are further highlighted in the relevant manage­ment sections below.

Conventional managementWithout intervention, any form of thalassaemia (excluding carrier status) is a progressive disease with increased morbidity as the patient advances in age.46 Moreover, the availability of effective therapeutic options and improved patient survival could allow multiple morbidities to manifest with age and the quality of life of the patient to deteriorate.47,48 Adult and elderly patients (older than 40 years) with thalassaemia should also be considered at risk of diseases that are common in non­thalassaemic populations, such as cancer and cardiovascular disease. Management guidelines for both patients with TDT and, more recently, NTDT, are available as part of the global efforts of the Thalassaemia International Federation to improve the outcomes of patients affected by these disorders.23,33

TransfusionTransfusion therapy works by supplying normal erythrocytes and suppressing ineffective erythropoiesis, essentially controlling all downstream pathophysio­logical mechanisms in thalassaemia.49–51 Advances in transfusion and iron­chelation therapy were associated with improvements in survival in long­term follow­up studies evaluating different birth cohorts of patients with β­thalassaemia major.52,53 Blood product screening, preparation, and administration practices have largely improved over the past 3 decades,53,54 and nowadays most patients with TDT can successfully achieve target haemoglobin concentrations of 90–105 g/L (110–120 g/L in those with heart disease); although access to blood for transfusion therapy remains a challenge in resource­poor countries and poses a substantial public health burden.33 In patients with NTDT, however, the indicators for when blood transfusion is necessary are not as well established, although increased morbidity has been identified in patients with β­thalassaemia intermedia who have haemoglobin concentrations of less than 100 g/L.39 Transfusions are sporadically given to patients when under acute stress or experiencing a drop in haemoglobin concentration, such as during infections, pregnancy, or surgery. Some patients are maintained on a regular transfusion programme following these incidents—a practice that in many cases might not be clinically indicated. Haemoglobin concentrations in isolation should not be an indication for lifelong, regular transfusions in patients with NTDT, especially given that many patients can adapt to their chronic anaemia without substantial bone marrow stress.55,56 Nonetheless, a role for regular transfusion therapy for a period of months or years has been established in patients with NTDT. Although data have emerged from obser­vational studies reporting improved growth variables in children, and a lower proportion of morbidities in regularly transfused adults than in those not regularly transfused, especially in patients with β­thalassaemia intermedia patients, including leg ulcers, throm­botic events, pulmonary hypertension, silent strokes, and extramedullary­ haemopoietic pseudo tumours.23,34,55–61 Collectively, these data might explain the differential clinical picture and increased proportion of such mor­bidities in patients with NTDT than in those with TDT.24,25,35 Transfusion therapy, however, does not come without its own side­effects. Although the risks of alloimmunisation and blood­borne infection remain a concern in transfused patients, the greatest challenge with regular transfusion therapy is secondary­iron overload.23,33

The human body has no means to excrete iron. Transfused blood contains around 200–250 mg of iron per unit. Patients receiving 2–4 units of blood per month will have an annual accumulation of 5000–10 000 mg of iron or 0·3–0·6 mg/kg per day.33,62 With continued transfusions, the reticuloendothelial system—primarily in the liver—

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can no longer retain all the extra iron. Iron then enters the plasma in amounts that exceed the transport capacity of circulating transferrin. Consequentially, non­transferrin­bound iron exists in the plasma as a heterogeneous mixture of iron complexes that appear to be the major mediators of hepatic and extrahepatic tissue damage in transfusional iron overload.63 Non­transferrin­bound iron enters specific cells, particularly hepatocytes, cardio­myocytes, anterior pituitary cells, and pancreatic β cells. In these cells iron accumulation leads to the generation of reactive­oxygen species, resulting in damage to lipids, proteins, DNA, and subcellular organelles, including lysosomes and mitochondria. This injury can result in cellular dysfunction, apoptosis, and necrosis that translates into target organ toxicity and dysfunction.63 The clinical burden of iron overload and its management are further illustrated in the following section.

Iron chelationWhether iron overload develops from increased intestinal absorption—such as in patients with NTDT—or is secondary to regular transfusions, it can cause substantial morbidity and mortality, and so warrants prompt diag­nosis and effective management. In patients with TDT, iron accumulation in organ tissues is evident in children aged as young as 2–6 years,64–67 with cumulative iron overload subsequently leading to organ toxic­effects and dysfunction, for instance in the heart, liver, or endocrine glands.33 Cardiomyopathy has been the leading cause of death in patients with TDT,33,52 although more recently large cohort studies68 have also highlighted the high incidence of mortality from hepatic causes in patients with TDT. Iron overload in patients with NTDT is a cumulative process with advancing age, and concerns over secondary morbidities start beyond the ages of 10–15 years.24,46,69,70 Although cardiac iron overload is commonly absent in patients with NTDT,71–74 an association between iron overload and hepatic fibrosis has been observed in patients with β­thalassaemia intermedia,75 and the occurrence of hepatocellular carcinoma is increasingly being reported.76 Cumulative iron overload in patients with NTDT also increases their risk of developing thrombosis, pulmonary hypertension, silent strokes, hypo thyroidism, hypogonadism, osteo porosis, and renal disease, as is evident from studies in patients with β­thalassaemia intermedia.61,69,77–81

Several methods are currently available for the diagnosis and monitoring of iron overload in patients with TDT and NTDT. Serum ferritin assessment is widely available and might be the only assessment that is affordable in resource­poor countries. The assessment of serial serum ferritin concentrations can be a good indicator of iron chelator effectiveness,33 and available guidelines recom­mend measurement of serum ferritin concen trations every 3 months.23,33,82 Serum ferritin thresholds are commonly used to indicate the need for initiation or modification of iron­chelation therapy. In patients with

TDT, maintaining concentrations lower than 1000 μg/L of ferritin is associated with lower morbidity and mortality, and this threshold is most commonly used to indicate the need for initiation of iron­chelation therapy.33,52,83,84 Serum ferritin concentrations consistently higher than 2500 μg/L are associated with an increased risk of cardiac and endocrine disease, and thus are commonly used to flag the need for dose optimisation or a change in iron­chelation therapy.33,83,84 However, although the association of high serum ferritin concentrations with hepatic siderosis is usually clear, a study85 using imaging tech­niques to evaluate organ­specific iron overload failed to establish an association between serum ferritin concen­trations and cardiac siderosis. Moreover, single measure­ments of serum ferritin concentration are prone to mis interpretation or not being representative of a patient’s condition, considering their high variability when a patient is experiencing inflammation, infection, vitamin C deficiency, hepatic dysfunction, and severe iron overload with values higher than 4000 μg/L.33,86 This high variability suggests that, when available and affordable, the use of imaging to evaluate organ­specific siderosis should be recommended.33 Worth mentioning is that several case reports on patients with NTDT have confirmed that serum ferritin concentrations are commonly lower than in patients with TDT with the same hepatic iron concen­tration.87 In patients with NTDT (patients with β­ thalassaemia intermedia), concentrations of greater than 800 μg/L are associated with an increased risk of morbidity, and concentrations of less than 300 μg/L are associated with an absence of risk of morbidity. These thresholds are thus used to indicate the need for iron chelation initiation or interruption.23,69

Measurement of liver iron concentration has become common practice since the early 2000s, considering the strong correlation with total­body iron stores.88 Initial reports relied primarily on the use of liver biopsy, although the technique is now used less frequently and only reserved for those situations when assessment of hepatic histology is needed.89 The use of a Superconducting Quantum Interference Device (SQUID) is also still practised, but the technology is only available in few centres worldwide.33,90 The introduction of MRI for the non­invasive assessment of liver iron concentration, and later myocardial iron concentration, is considered one of the most important advances in thalassaemia care over the past decade. The use of MRI in clinical practice can allow better tailoring of iron­chelation therapy. For measurement of liver iron concentration, both the R2 and T2* techniques are used as they have been validated against liver biopsy measurements of iron.91,92 They are internationally reproducible and accurately measure liver iron concentration throughout the clinically relevant range with appropriate calibration and MRI acquisition techniques, and give a measurement of liver iron concentration in mg of iron per g of dry liver weight.93,94 They are also equally effective in evaluating chronic

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response to iron chelation.95,96 R2 MRI has also been validated in patients at different stages of liver fibrosis and grades of liver inflammation and the technique (FerriScan®, Resonance Health, Burswood, WA, Australia) is approved by the US Food and Drug Administration.94 Cardiac T2* MRI is used for the assessment of cardiac iron (values in ms), with recent validation against myocardial iron concentration in mg of iron per g of dry heart weight from post­mortem cardiac biopsies.97 The use of both techniques to evaluate iron content in other organs—such as the endocrine gland—has also been investigated, although they are less commonly used in routine practice.98–100 Certain liver iron concentrations and cardiac T2* thresholds have been linked to morbidity in patients with thalassaemia. In patients with TDT, liver iron concentrations greater than 7 mg/g are usually used to indicate increased risks of complication, and lower target concentrations are used to prevent iron­related complications, although this evidence has mainly been extrapolated from patients with here­ditary haemochromatosis.101–103 Concentrations greater than 15 mg/g are associated with progressive liver fibrosis and cardiac mortality. Cardiac T2* values of less than 20 ms are indicative of a declining left ventricular ejection

fraction and associated with arrhythmias, whereas values of less than 10 ms are associated with clinical heart failure and mortality.104–106 Thus, these thresholds are commonly used to determine a patient’s iron­overload profile, to evaluate their response to iron­chelation therapy in clinical trials, and to tailor their chelator type and dose in routine practice.33,107 In patients with NTDT (β­thalassaemia inter­media), liver iron concentration values higher than 5 mg/g are associated with increased morbidity,78 supporting the recommendation of iron­chelation initiation in patients showing liver iron concentrations greater than 5 mg/g.23 It should be noted that patients with α­thalassaemia do tend to show slower rates of iron overload than patients with other NTDT forms, and lower­intensity iron chelation standards are usually considered. Most available guide­lines recommend assessment every 6, 12, or 24 months depending on iron­overload severity and iron­chelation adjustment needs.23,33,82

Three iron chelators are currently available for the treatment of iron overload in patients with thalassaemia: deferoxamine in subcutaneous or intravenous injection, oral deferiprone in tablet or solution form, and oral deferasirox in dispersible tablet and, more recently, film­coated tablet forms (table 1), and guidelines for their use

Deferoxamine Deferiprone Deferasirox

Administration33

Method Subcutaneous or intravenous Oral Oral

Frequency 8–12 h, 5–7 days per week 3 times daily Once daily

Half-life of iron-free drug33 20–30 min 3–4 h 12–16 h

Lipid solubility33 Low Intermediate High

Route of iron excretion33 Urinary and faecal Urinary Faecal

Recommended dose23,33 30–60 mg/kg per day 75–100 mg/kg per day TDT: 20–40 mg/kg per day; NTDT: 5–20 mg/kg per day

TIF guidelines indication

TDT33 >2 years: first-line 2–6 years: no sufficient data; >6 years: second-line† 2–6 years: first-line (USA), second-line (EU); >6 years: first-line

NTDT23 No sufficient data No sufficient data >10 years: first-line

Most relevant clinical data

TDT33 Reduction in serum ferritin and liver iron concentration;108 improvement in cardiac T2*;108 improvement in cardiac dysfunction with continuous infusion109

Improvement of cardiac T2* in monotherapy or in combination with deferoxamine (higher doses than commonly used in clinical practice)‡;110,111 improvement in cardiac dysfunction in combination with deferoxamine†‡;112 improvement in endocrine dysfunction in combination with deferoxamine or deferasirox113,114

Reduction in serum ferritin and liver iron concentration after up to 5 years, and cardiac T2* after up to 3 years of therapy, even in patients with severe iron overload;115–118 not inferior to deferoxamine for improving cardiac T2*;108 improvements in hepatic fibrosis and inflammation;119 stabilisation of heart function;108,115 stabilisation of endocrine function120

NTDT23 Data restricted to case series and small studies

Data restricted to case series and small studies Significant reduction in serum ferritin and liver iron concentration after up to 2 years of therapy121

Main adverse events33 Ocular and auditory symptoms, bone-growth retardation, local reactions, allergy

Gastrointestinal symptoms, arthralgia, agranulocytosis or neutropenia

Gastrointestinal symptoms, increased creatinine, increased hepatic enzymes

Pregnancy33 Contraindicated (but has been used in third trimester)

Contraindicated Contraindicated

TDT=transfusion-dependent thalassaemia. NTDT=non-transfusion-dependent thalassaemia. TIF=Thalassaemia International Federation. †In a recent Cochrane review122 it was concluded that in the absence of data from randomised controlled trials, there is no evidence to suggest the need for a change in the current treatment recommendations. Specifically, deferiprone is indicated for treating iron overload in patients with thalassaemia major when deferoxamine is contraindicated or inadequate. Intensified deferoxamine treatment, use of other oral iron chelators, or both, remains the established treatment to reverse cardiac dysfunction due to iron overload. ‡Despite results of some trials110,111 indicating the superiority of deferiprone, or its combination with deferoxamine, compared with deferoxamine monotherapy in improving cardiac T2*, a meta-analysis123 concluded that deferoxamine and deferiprone monotherapy appeared to reduce myocardial iron by similar amounts. Moreover, the addition of deferiprone to deferoxamine in a recent study124 did not enhance its ability to improve cardiac dysfunction compared to monotherapy. There is an urgent need for high-quality trials to compare the overall clinical efficacy and long-term outcome of treatment with deferiprone combined with deferoxamine.122

Table 1: Characteristics and evidence on iron chelators for the management of iron overload in thalassaemia

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are now widely available.23,33,82 Data from several large, randomised trials since the development of MRI technology showed the efficacy and safety of oral­iron­chelation therapy in removing iron from the liver and heart, which represents a considerable advance in patient management owing to the greater convenience compared to parenteral deferoxamine. However, parenteral deferox­amine remains the treatment of choice in patients with decompensated heart disease, and might be the only affordable option in resource­poor countries.33 None­theless, the search for other novel iron chelators that can address the specific efficacy or safety shortcomings of these chelators continues.33 Moreover, successful manage ment of iron overload extends beyond chelator efficacy and safety. Ensuring adherence to treatment is essential, and applicable to most chelators irrespective of their administrative form. Adherence to iron­chelation therapy correlates with both effective management and patient survival, as is evident from several studies.125,126 Moreover, appropriate dose determination and adjust­ment of the iron chelator according to baseline and ongoing iron intake is essential to ensure adequate response and avoid overchelation.33

SplenectomySplenectomies have traditionally been performed as an alternate or adjunct to transfusion therapy. Although some studies have found that splenectomies have resulted in improvements in growth, quality of life, and haemoglobin concentration for some patients with haemoglobin E/β­thalassaemia.127 The procedure has now become almost obsolete in patients with TDT, especially in the context of improved access to and safety of blood transfusions with regards to blood­borne infections. The procedure is still more commonly used in patients with NTDT, because it might be enough to improve mild­to­moderate anaemia and avoid the need for transfusions.25,31,34,57,58 However, data for serious adverse events caused by splenectomies continue to accumulate in the thalassaemia patient population, alongside the known risk of infection and sepsis. Removal of the spleen promotes a hypercoagulable state in thalassaemia because of the decreased ability to scavenge procoagulant red blood cells and activated platelets. Observational studies have further established that splenectomised NTDT patients are at a 4–5 fold increased risk of overt venous thrombosis and other vascular manifestations like pulmonary hypertension, leg ulcers, and silent strokes than non­splenectomised patients.60,61 In patients with β­thalassaemia intermedia, the long­term risk of thrombosis is further increased in those with concomitant high counts of nucleated red blood cells (≥300 × 10⁶ cells per L) and platelets (≥500 × 10⁹ platelets per L), and those patients who never received any transfusions.128 The spleen also usually acts as a reservoir for scavengers of toxic iron in the body, and so removal of the spleen could also decrease the ability of these scavengers, thus putting patients at increased risk of end­organ damage.60 The current recommendations for splenectomy are restricted to those patients with an inability to receive transfusion and iron­chelation therapy, as well as those with clinically symptomatic spleno­megaly or hyper splenism.23,33 When indicated, appro­priate treatment with vaccination and antibiotics is recommended, and prophylaxis with aspirin or low­molecular weight heparins for high­risk patients.23,33

Management of specific morbiditiesPrevention of morbidity in patients with thalassaemia starts with appropriate and adequate application of conventional therapies including transfusion or iron chelation, or both.23,33 Special clinical considerations and management options beyond conventional therapy are summarised in table 2 for key clinical complications.

Haemopoietic stem-cell transplantationReplacement of mutant haemopoietic cells using haemopoietic stem­cell transplantation is the only existing curative therapy for thalassaemia and is now an established approach to correct defective erythropoiesis, particularly when matched sibling donors are available.

Assessment considerations (indication) Management considerations

Cardiac dysfunction and arrhythmia

Echocardiography (routine), electrocardiogram (routine)

As per standard care

Pulmonary hypertension

Tricuspid regurgitant jet velocity (routine), right-heart catheterisation (high risk)

As per standard care, sildenafil citrate, bosentan

Cerebrovascular events

MRI and MRA (high risk) As per standard care, antiplatelet prophylaxis

Venous thrombosis Standard imaging (suggestive of signs and symptoms)

Anticoagulant therapy, medical and surgical prophylaxis

Leg ulcers Physical examination (routine) Topical measures, pentoxifylline, hydroxycarbamide, hyperoxygenation

Viral hepatitis Viral serology (routine in transfused patients), viral RNA-PCR (if positive serology)

Hepatitis B vaccination, antiviral therapy

Hepatic fibrosis, cirrhosis, and cancer

Live function tests (routine), ultrasound (high risk), α-fetoprotein (high risk), transient elastography (investigational)

As per standard care

Endocrine disease Growth retardation (routine), sexual development (routine), endocrine-function tests (routine), bone-mineral density (routine)

As per standard care

Bone disease Bone-mineral density (routine) As per standard care, bisphosphonates

Pregnancy As per high-risk pregnancy Revisit iron chelation, anticoagulation prophylaxis, maintenance of haemoglobin concentration and heart function

Extramedullary haematopoietic pseudotumours

Physical exam and imaging to rule out compression (suggestive signs and symptoms)

Hypertransfusion, radiation, surgery

Haemolytic crisis (haemoglobin H disease)

Infection screening, electrolytes Adequate hydration, correction of blood electrolytes, control body temperature, antibiotics or antivirals

The frequency and age at the start of assessment should rely on the underlying diagnosis and individual patient’s needs. Further management details are available in the respective Thalassaemia International Federation guidelines.23,33 MRA=magnetic resonance angiography.

Table 2: Management considerations for specific morbidities in thalassaemia patients beyond transfusion and iron chelation

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Haemopoietic stem­cell transplantation is now widely applied with a disease­free survival exceeding 80% with transplants from HLA­matched­sibling donors.129 Moreover, improvements in the management of graft­versus­host disease and inducing graft tolerance have encouraged the use of unrelated donors and umbilical­cord blood as the haemopoietic stem­cell source for patients who do not have a matched­sibling donor. Nevertheless, matched­unrelated transplants for high­risk patients with thalassaemia have an overall survival of 65%. However, a 5–10% mortality from transplant conditioning, graft­versus­host disease, and graft failure continues to limit the acceptability of this treatment method. The need for complete myeloablation, which can result in infertility and other toxic­effects, is also a concern, although improved approaches with reduced­intensity conditioning are being developed.130 Clinical guidelines specific to patients with thalassaemia are now available.129

Future outlookIn the past decade several promising targets and associated therapeutic options have emerged for patients with thalassaemia, though primarily for those with β­thalassaemia, which is reasonable considering the patient’s more complex and advanced management needs (figure 3).6,42,43 These therapeutic options can be classified into three major categories on the basis of their attempts to address different aspects of the underlying pathophysiology of thalassaemia: correction of globin­chain imbalance, addressing ineffective erythro poiesis, and improving how the body handles iron. In this section, we will shortly discuss some of the emerging approaches in each of these areas.

Gene therapy is based on the idea that if there is a defect in the production of β­globin in β­thalassaemia, exo­genous production of β­like globins could correct the disorder. The haemopoietic system is convenient for such approaches, since haemopoietic stem cells from an individual with β­thalassaemia can be isolated and trans­duced with viruses to introduce exogenous genetic

material, such as β­like globin transgenes that can allow for such exogenous gene expression. Following successful application in a few patients with thalassaemia,131 clinical trials have been initiated.

Several approaches are also being developed to address the α­globin or β­globin chain imbalance in patients with thalassaemia by stimulation of γ­globin production.26 Although many small trials have examined the use of currently available cytotoxic agents, such as hydroxy­carbamide, there is a scarcity of published results and studies.132 The limitations of the currently available approaches to address the α­globin or β­globin chain imbalance have motivated a considerable amount of research into better understanding the molecular regulation of γ­globin. In recent years, human­genetic studies have provided important insights into this area and have suggested several promising molecular targets, such as BCL11A, which is a transcription factor capable of silencing the γ­globin genes specifically without affecting production of red blood cells.26 Instead of gene therapy, recent approaches have been developed to directly correct genetic mutations in the endogenous DNA of the cell or to disrupt specific DNA sequences in the genome. This approach is known as genome editing and has been facilitated through the identification of several enzymes, including CRISPR/Cas9, that can intro­duce DNA breaks in specific regions of the genome. Although it is challenging to envision correcting every one of the hundreds–thousands of mutations that cause β­thalassaemia, disruption of factors that silence the γ­globin genes, such as BCL11A, might allow more immediate treatment of β­thalassaemia with current genome editing approaches.6

Ineffective erythropoiesis is the major contributor to the anaemia observed in patients with β­thalassaemia. In the past few years, a group of ligand traps (which include sotatercept and luspatercept) have been developed that have shown potent stimulation of late­stage erythropoiesis and improved ineffective erythropoiesis in patients with β­thalassaemia.6,133,134 Ongoing clinical studies of these drugs are evaluating their role in reducing transfusion

Figure 3: Novel targets and therapies for patients with β-thalassaemiaEPO=erythropoietin. ROS=reactive-oxygen species.

α/β-Chain imbalance Ineffective erythropoiesis

Minihepcidins

JAK2 inhibitors

SotaterceptLuspatercept

Gene therapyGene editing

TMPRSS inhibitors

TMPRSS6↓ Differentiation ↓ Hepcidin

↑ Proliferation

↑ ROS

↑ EPO

↑ GDF11

Anaemia

Iron overload

Erythroferrone

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requirements and raising haemoglobin concentrations in patients with TDT and NTDT, respectively. JAK2 inhibition, an established therapy in patients with myeloproliferative disorders, can also ameliorate ineffective erythropoiesis and decrease spleen size in mice with thalassaemia, which has motivated ongoing clinical trials in this area to evaluate the role of JAK2 in patients with TDT.43

In NTDT mouse models, moderate transgenic hepcidin expression decreased iron loading in the liver, and resulted in an extended red blood cell lifespan, increased haemoglobin concentrations and amelioration of splenomegaly.43,135 Recent preclinical studies43,136 have suggested that long­acting hepcidin analogues (mini­hepcidins) could be beneficial to restrict iron absorption and use in the setting of iron overload, with beneficial effects on ineffective erythropoiesis. If such molecules are developed for clinical use, these could help to further restrict iron overload either alone or in combination with currently available iron chelators. An alternative approach is to stimulate endogenous hepcidin prod uction through the downregulation of a metallo protease, TMPRSS6, that plays a key role in hepcidin expression.137 Antisense oligonucleotides and siRNAs targeting TMPRSS6 have been effectively used to stimulate hepcidin, reduce the iron burden, and improve in effective erythropoiesis and red blood cell survival in preclinical iron­overload models138,139—clinical trials are expected soon.

Concluding remarksOur improved understanding of the pathophysiology and disease burden in patients with thalassaemia has helped optimise disease management and construct a plan for the development of novel therapeutics. With several treatment options available, the need for comparative and combinative clinical trials is essential to design the best management approach. Nonetheless, the complexity of thalassaemia as a disease might always imply the need for individualised therapy, and offering the right treatment approach to the right patient. The equal prioritisation of efforts to promote the prevention of thalassaemia births through nationwide screening programmes and pre­marital policy adaptations should also be addressed. Successful examples of a remarkable decline in disease incidence from the Mediterranean should aim to be reproduced in resource­poor countries where the disease continues to pose public health concerns. This effort, however, will require challenges attributed to economic, cultural, and religious differences to be addressed with innovative approaches to premarital and prenatal coun­selling. Expanded­education pro grammes and health­care­system awareness are also needed nowadays in high­income communities where thalassaemias were, until recently, not commonly present. Lastly, collaborations and partnerships between centres for the exchange of expertise and resources could help improve the diagnosis and management of the thalassaemias, especially in low­income countries where they are most common.

ContributorsATT, DJW, and MDC participated in the literature search, manuscript drafting, and review. All authors approved the final manuscript before submission.

Declaration of interestsATT and MDC report grants and personal fees from Novartis and Celgene, outside the submitted work. DJW declares no competing interests.

AcknowledgmentsWe thank Khaled Musallam and Vijay Sankaran for their constructive review of this manuscript. We apologise to those authors whose work could not be cited in this review because of space limitations.

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