MED ICA L PROGR ES S

Review Article

Medical Progress T HE b-T HALASSEMIAS

NANCY F. OLIVIERI, M.D.

N 1925, Thomas Cooley and Pearl Lee described a form of severe anemia, occurring in children of Italian origin and associated with splenomegaly and characteristic bone changes.1 Over the next decade, a milder form was described independently by several Italian investigators.2-4 Because all early cases were reported in children of Mediterranean origin, the disease was later termed thalassemia, from the Greek word for sea, thalassa.5 Over the next 20 years, it became apparent that Cooley and Lee had described the homozygous or compound heterozygous state for a recessive mendelian disorder not confined to the Mediterranean, but occurring widely throughout tropical countries. In the past 20 years, the two important forms of this disorder, a- and b-thalassemia, resulting from the defective synthesis of the a- and b-globin chains of hemoglobin, respectively, have become recognized as the most common monogenic diseases in humans.6 This article focuses on the b-thalassemias, the severe forms of which are by far the most important of all the thalassemias. The molecular and clinical aspects of the severe a-thalassemia syndromes have been reviewed elsewhere.7,8
DISTRIBUTION AND POPULATION AT RISK

length polymorphisms, or haplotypes, within the b-globin cluster. A limited number of haplotypes are found in each population, so that 80 percent of the mutations are associated with only 20 different haplotypes. This observation has helped demonstrate the independent origin of b-thalassemia in several populations.10 There is evidence that the high frequency of b-thalassemia throughout the tropics reflects an advantage of heterozygotes against Plasmodium falciparum malaria,11 as has already been demonstrated in a-thalassemia.12
MOLECULAR PATHOLOGY
Structure and Synthesis of Hemoglobin

The structure and regulation of the human globin genes have been reviewed elsewhere13; only aspects with direct relevance to an understanding of the molecular pathology of the b-thalassemias are outlined here. The b-like globin genes, a linked cluster on chromosome 11, are arranged over approximately 60,000 nucleotide bases (Fig. 1). Promoter elements upstream from the initiation codon of each active gene are involved in the initiation of transcription. The cluster also contains other regulatory elements that interact to promote erythroid-specific gene expression and to coordinate the developmental regulation of each gene.
Hemoglobin Switching

The b-thalassemias are widespread throughout the Mediterranean region, Africa, the Middle East, the Indian subcontinent and Burma, Southeast Asia including southern China, the Malay Peninsula, and Indonesia. Estimates of gene frequencies range from 3 to 10 percent in some areas.9 Within each population at risk for b-thalassemia a small number of common mutations are found, as well as rarer ones; each mutation is in strong linkage disequilibrium with specific arrangements of restriction-fragment

As an adaptation to changing oxygen requirements, different hemoglobins, all composed of two different pairs of globin chains each attached to a heme moiety, are synthesized in the embryo, fetus, and adult.14 Severe b-thalassemia usually becomes manifest as a result of the decline in the synthesis of fetal hemoglobin (a2g 2) during the first year of life (Fig. 1). The precise mechanisms that control the switch from the production of fetal hemoglobin to that of adult hemoglobin (a2 b 2) (Fig. 1) are not fully understood.13-16
Mutations Causing b-Thalassemia

From the University of Toronto, Toronto. Address reprint requests to Dr. Olivieri at the Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8, Canada, or at noliv@sickkids.on.ca. 1999, Massachusetts Medical Society.

Nearly 200 different mutations have been described in patients with b-thalassemia and related disorders. Although most are small nucleotide substitutions within the cluster, deletions may also cause b-thalassemia.9 All the mutations result in either the absence of the synthesis of b-globin chains (b0-thalassemia) or a reduction in synthesis (b+-thalassemia) (Fig. 2). Mutations in or close to the conserved promoter sequences and in the 5' untranslated region downVol ume 341 Numb e r 2

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Chromosome 11

Locus-control region

cb

50

Globin-Chain Synthesis (% of total)

a g b

30

10

z d

18

30

18

30

42

Before Birth (wk)

Birth

After Birth (wk)

Figure 1. The b -Globin Gene Cluster on the Short Arm of Chromosome 11. In Panel A, the b -globinlike genes are arranged in the order in which they are expressed during development. The Gg and Ag genes are both active genes that produce g -globin chains that differ only at position 136 (glycine is the product of the Gg gene, and alanine is the product of the Ag gene). The cb gene is a pseudogene, an evolutionary remnant of a previously active b -globinlike gene. Areas of nucleotide homology upstream from the initiation codon of each active gene, termed promoter elements, are involved in the initiation of transcription and hence play a vital part in gene regulation. The cluster also contains regulatory elements that interact to promote erythroid-specific gene expression and to coordinate the developmental regulation of each gene, including hemoglobin switching. These include enhancers, distant regulatory elements that increase gene expression, and a master sequence, the clusters essential distal regulatory element, the b -globin locus-control region. This is a region that lies 20 kb upstream from the e-globin gene.13 It encompasses five erythroid-lineagespecific nuclease hypersensitive sites (shown in red) that permit expression of the downstream genes. In addition to these elements for up-regulation, several suppressor regions, or silencers, have been defined in the b -globin gene cluster. Panel B shows the timing of the normal developmental switching of human hemoglobin. Early in fetal life the synthesis of the embryonic a-globinlike (z) chains switches to that of a-globin, which is produced thereafter. At the same time, the synthesis of embryonic beta-like (e) chains switches to that of g -globin chains. The a-globin and g -globin chains combine to form fetal hemoglobin (a2g2), the main b -globinlike globin during the remainder of fetal life and throughout early postnatal life. As a result of the decline in the synthesis of g -globin chains in patients with b -thalassemia, fetal hemoglobin production becomes insufficient to compensate for the excess of a-globin chains, the production of which is unaffected in b -thalassemia.

regulate transcription, usually resulting in mild b+-thalassemia. Transcription is also affected by deletions in the 5' region, which completely inactivate transcription and result in b0-thalassemia. Both splicing of the messenger RNA (mRNA) precursor and ineffective cleavage of the mRNA tran100
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script result in b-thalassemia. In some mutations, no normal message is produced, whereas other mutations only slightly reduce the amount of normally spliced mRNA. Mutations within invariant dinucleotides at intronexon junctions, critical to the removal of intervening sequences and the splicing of exons

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Deletions

Intron Intron 5' Exon 1 Intron Exon 2 Exon 3 Intron 3'

Other Mutations
Mutation affecting initiation of transcription Mutation affecting splicing of RNA from introns Polyadenylation-signal mutation Mutation affecting initiation of translation Nonsense mutation Frame-shift mutation

Figure 2. The Normal Structure of the b-Globin Gene and the Locations and Types of Mutations Resulting in b-Thalassemia. All b-globinlike genes contain three exons and two introns between codons 30 and 31 and 104 and 105, respectively. The primary action of all the mutations is to abolish the output of b-globin chains (b0-thalassemia; shown in red) or reduce the output (b+-thalassemia; shown in green). The 170 different mutations that act in this way may interfere with the action of the b-globin gene at the transcriptional level, in the processing of the primary transcript, in the translation of b-globin messenger RNA, or in the post-translational stability of the b-globin gene product.

to produce functional mRNA, result in b0-thalassemia. Mutations in highly conserved nucleotides flanking these sequences, or in cryptic splice sites, which resemble a donor or acceptor splice site, result in severe as well as mild b+-thalassemia. Substitutions or small deletions affecting the conserved AATAAA sequence in the 3' untranslated region result in ineffective cleavage of the mRNA transcript and cause mild b+-thalassemia. Mutations that interfere with translation involve the initiation, elongation, or termination of globin-chain production and result in b0-thalassemia. Approximately half of all b-thalassemia mutations interfere with translation; these include frame-shift or nonsense mutations, which introduce premature termination codons and result in b0-thalassemia. A more recently identified family of mutations, usually involving exon 3, results in the production of unstable globin chains of varying lengths that, together with a relative excess of a-globin chains, precipitate in red-cell precursors and lead to ineffective erythropoiesis, even in the heterozygous state. This is the molecular basis for dominantly inherited (b+) thalassemia. In addition,

missense mutations, resulting in the synthesis of unstable b-globin chains, cause b-thalassemia.
PATHOPHYSIOLOGY
Mechanisms of Anemia

In severe untreated b-thalassemia, erythropoiesis may be increased by a factor of up to 10, more than 95 percent of which may be ineffective. Ineffective erythropoiesis, the hallmark of b-thalassemia, is a result of the myriad deleterious effects of a relative excess of a-globin chains.17 This relative excess interferes with most stages of normal erythroid maturation: both intramedullary death of red-cell precursors through arrest in the G1 phase of the cell cycle18 and accelerated intramedullary apoptosis of late erythroblasts19,20 have been demonstrated. Studies of the consequences of the accumulation of excess a-globin chains and their degradation products within the redcell membrane and its skeleton20-22 have also demonstrated abnormalities in the ratio of spectrin to band 3 and in the function of band 4.1. This subject has been thoroughly reviewed recently.20 The observaVol ume 341 Numb e r 2

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Excess free a-globin chains Denaturation Degradation

Formation of heme and hemichromes

Iron-mediated toxicity

Hemolysis

Ineffective erythropoiesis

Membrane binding of IgG and C3

Removal of damaged red cells

Increased erythropoietin synthesis

Reduced tissue oxygenation

Anemia

Splenomegaly

Skeletal deformities, osteopenia

Erythroid marrow expansion

Increased iron absorption

Iron overload

Figure 3. Effects of Excess Production of Free a-Globin Chains. Primary disease processes are indicated in orange, and compensatory mechanisms in yellow. Excess unbound a-globin chains and their degradation products precipitate in red-cell precursors, causing defective maturation and ineffective erythropoiesis. Hemolysis, owing to the presence of inclusions in the red cells and damage to their membranes by a-globin chains and their degradation products, also contributes to the anemia. Anemia stimulates the synthesis of erythropoietin, leading to an intense proliferation of the ineffective marrow, which in turn causes skeletal deformities and a variety of growth and metabolic abnormalities. The anemia is further exacerbated by hemodilution, caused by the shunting of blood through the expanded marrow, and by splenomegaly resulting from entrapment of abnormal red cells in the spleen. Bone marrow expansion also results in characteristic deformities of the skull and face, severe osteopenia, and increased iron absorption.

tion that the presence of excess membrane iron may aggravate membrane changes 22 has led to interest in the red-cell membrane as a potential therapeutic target in b-thalassemia. In a mouse model, increased cellular rigidity and decreased stability in connection with membrane-associated a-globin chains 23 have reportedly been ameliorated during exposure to agents that bind membrane iron.24 Further understanding of these processes may guide future therapies.
Clinical Consequences of Anemia

progressive splenomegaly exacerbate anemia (Fig. 3). Increased erythropoietin synthesis may stimulate the formation of extramedullary erythropoietic tissue, primarily in the thorax and paraspinal region. Marrow expansion also results in characteristic deformities of the skull and face, as well as osteopenia and focal defects in bone mineralization,25,26 and may aggravate a painful periarticular syndrome characterized histologically by microfractures and osteomalacia.27 Marrow hyperplasia leads ultimately to increased iron absorption and progressive deposition of iron in tissues.
Cellular Heterogeneity and Fetal Hemoglobin Production

The severe ineffective erythropoiesis results in erythroid marrow expansion to as much as 30 times the normal level. Both an increase in plasma volume as a result of shunting through expanded marrow and
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Although fetal hemoglobin synthesis persists after birth to some degree, its production is insufficient

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to compensate for the reduced synthesis of b-globin chains and the relative excess of a-globin chains.15 The elevated concentrations of fetal hemoglobin 2 to 4 g per deciliter observed in patients with b-thalassemia reflect a combination of the selection of precursors that produce relatively more fetal hemoglobin and erythroid expansion, which appears to favor the production of g-globin chains. Even higher fetal hemoglobin concentrations are associated with specific b-thalassemia alleles 28,29 or other genetic determinants within or linked to the b-globin complex.15 At least two other determinants may affect the synthesis of fetal hemoglobin, one on chromosome 630 and the other on the X chromosome.31,32
Clinical Forms

regularly seen only in countries without resources to support long-term transfusion programs.
Relation between Genotype and Phenotype

The b-thalassemias include four clinical syndromes of increasing severity: two conditions are generally asymptomatic, the silent carrier state and b-thalassemia trait, and usually result from the inheritance of one mutant b-globin gene, and two require medical management, thalassemia intermedia and thalassemia major. The more severe forms most often result from homozygosity or compound heterozygosity for a mutant b-globin allele and, occasionally, from heterozygosity for dominant mutations.33 Homozygous or compound heterozygous b-thalassemia usually presents no diagnostic problems. The early onset of anemia, characteristic blood changes, and elevated fetal hemoglobin concentrations are found in no other condition. The diagnosis can be confirmed by the demonstration of the b-thalassemia trait in both parents. This condition is characterized by mild anemia, reduced mean cell volumes and mean cell hemoglobin concentrations,29 and elevated concentrations of the normal minor adult component of hemoglobin (usually exceeding 3.5 percent), hemoglobin A2 (a2d 2). Thalassemia major and thalassemia intermedia have no specific molecular correlate but encompass a wide spectrum of clinical and laboratory abnormalities.34 Patients referred to as having thalassemia major are usually those who come to medical attention in the first year of life and subsequently require regular transfusions to survive. Those who present later or who seldom need transfusions are said to have thalassemia intermedia.35 After thalassemia is diagnosed, patients who appear not to require immediate transfusion may benefit from a period of observation and folate repletion, particularly if the disease is diagnosed after the age of one year. This approach will allow the identification of patients in whom early growth and development are normal and whose well-compensated anemia may be exacerbated only by infection, folate deficiency, or increasing hypersplenism.34-37 With advancing age, even patients with mild forms may have serious complications, including osteopenia, iron loading in tissues, and ectopic marrow expansion. The classic changes of untreated thalassemia major are now

Several genetic factors may ameliorate the severity of b-thalassemia.29 First, the underlying mutations vary widely in their effect on the synthesis of b-globin chains.38 Co-inheritance of a-thalassemia may reduce the severity of the globin-chain imbalance. Many different interactions with structural hemoglobin variants may also result in a complex series of clinical phenotypes.29 The interactions of b-thalassemia with two of these variants, hemoglobin S and hemoglobin E, are of global importance. The clinical consequences of the interaction with hemoglobin S depend mainly on the b-thalassemia allele. If inherited with b0-thalassemia or severe b+-thalassemia, the resulting clinical disorder may be indistinguishable from sickle cell anemia. By contrast, interactions with mild b+-thalassemia alleles produce a milder sickling disorder. Although hemoglobin E b-thalassemia is probably the most common serious hemoglobinopathy worldwide,39 its natural history remains poorly understood.29 The mutation that produces hemoglobin E activates a cryptic splice site in exon 1 in the b-globin gene; hence, hemoglobin E is associated with mild b-thalassemia. For reasons that are not well understood,29 the interaction of hemoglobin E and b-thalassemia results in a wide spectrum of clinical disorders: some are indistinguishable from thalassemia major, and some are much milder and not transfusiondependent. Finally, a number of acquired and environmental factors, including progressive splenomegaly, exposure to infections, socioeconomic factors, and the availability of medical care, may also modify the severity of the disease.
COMPLICATIONS OF DISEASE
Iron Overload

Iron overload of tissue, which is fatal with or without transfusion if not prevented or adequately treated, is the most important complication of b-thalassemia and is a major focus of management.40 In patients who are not receiving transfusions, abnormally regulated iron absorption results in increases in body iron burden ranging from 2 to 5 g per year, depending on the severity of erythroid expansion.41,42 Regular transfusions may double this rate of iron accumulation. Although most clinical manifestations of iron loading do not appear until the second decade of life in patients with inadequate chelation, evidence from serial liver biopsies in very young patients indicates that the deleterious effects of iron are initiated much earlier than this. After approximately one year of transfusions, iron begins to be deposited in parenchymal tissues,43 where it may cause substantial toxicity as compared with that within reticVol ume 341 Numb e r 2

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uloendothelial cells.44,45 As iron loading progresses, the capacity of serum transferrin, the main transport protein of iron, to bind and detoxify iron may be exceeded and a nontransferrin-bound fraction of plasma iron may promote the generation of free hydroxyl radicals, propagators of oxygen-related damage.44,45 The advances in free-radical chemistry that have clarified the toxic properties of these and other oxygen-derived species generated by iron, which may cause widespread tissue damage, have recently been summarized.45 Although the body maintains a number of antioxidant mechanisms against damage induced by free radicals, including superoxide dismutases, catalase, and glutathione peroxidase, in patients with large iron burdens these may not prevent oxidative damage.44,45 In the absence of chelating therapy the accumulation of iron results in progressive dysfunction of the heart, liver, and endocrine glands.40 Within the heart, changes associated with chronic anemia are usually present in patients who are not receiving transfusions and are aggravated by iron deposition. In response to iron loading, human myocytes in vitro increase the transport of nontransferrin-bound iron,46 possibly thereby aggravating cardiac iron loading. Extensive iron deposits are associated with cardiac hypertrophy and dilatation, degeneration of myocardial fibers, and in rare cases fibrosis.47 In patients who are receiving transfusions but not chelating therapy, symptomatic cardiac disease has been reported within 10 years after the start of transfusions48 and may be aggravated by myocarditis49 and pulmonary hypertension.50,51 The survival of patients with b-thalassemia is determined by the magnitude of iron loading within the heart.52,53 Iron-induced liver disease is a common cause of death in older patients54 and is often aggravated by infection with hepatitis C virus. Within two years after the start of transfusions, collagen formation55 and portal fibrosis56 have been reported; in the absence of chelating therapy, cirrhosis may develop in the first decade of life.43,57,58 The extent of these processes may be underestimated if fewer than three cores of liver are sampled at biopsy.59 The risk of hepatic fibrosis is augmented at body iron burdens corresponding to hepatic iron concentrations of more than 7 mg per gram of liver, dry weight (Fig. 4).61 As in cultured heart cells, in cultured hepatocytes the transport of nontransferrin-bound iron is increased,62 possibly aggravating iron loading in vivo. The striking increases in survival in patients with b-thalassemia over the past decade have focused attention on abnormal endocrine function, now the most prevalent iron-induced complication in older patients. Iron loading within the anterior pituitary is the primary cause of disturbed sexual maturation, reported in 50 percent of both boys and girls with the condition.63 Furthermore, early secondary amenor104
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rhea occurs in approximately one quarter of female patients over the age of 15 years.63 Even in the modern era of iron-chelating therapy, diabetes mellitus is observed in about 5 percent of adults.63 As the iron burden increases and iron-related liver dysfunction progresses, hyperinsulinemia occurs as a result of reduced extraction of insulin by the liver, leading to exhaustion of beta cells and reduced circulating insulin concentrations.64 Studies reporting reduced serum concentrations of trypsin and lipase65 suggest that the exocrine pancreas is also damaged by iron loading. Over the long term, iron deposition also damages the thyroid, parathyroid, and adrenal glands66,67 and may provoke pulmonary hypertension, right ventricular dilatation, and restrictive lung disease.51,68-70 In most studies, bone density is markedly reduced in patients with b-thalassemia, particularly those with hypogonadism. Osteopenia may be related to marrow expansion, even in patients who receive transfusions,42 or to iron-induced osteoblast dysfunction, diabetes, hypoparathyroidism, or hypogonadism.71-73
CONTROL AND MANAGEMENT
Prevention Programs and Prenatal Diagnosis

Screening programs, aimed at prevention of the disease, and prenatal diagnosis have resulted in a marked reduction in the birth rate of affected children in Greece, Cyprus, continental Italy, and Sardinia.74 Widespread use of similar programs in other areas of the world has not yet been possible. Screening for carriers is performed most efficiently by measurement of the red-cell indexes and, in samples from persons with reduced mean cell volumes and mean cell hemoglobin concentrations, estimation of the hemoglobin A2 concentration. The practical problems associated with screening for rarer forms of b-thalassemia and the effect of coexistent a-thalassemia on the red-cell indexes have been reviewed recently.29 Prenatal diagnosis, first carried out by fetal-blood sampling and assessment of globin-chain synthesis in fetal blood, more recently has involved direct analysis of fetal DNA obtained by chorionicvillus sampling. This approach is associated with a very slightly increased risk of fetal loss and an error rate in experienced laboratories of less than 1 percent. The practical aspects of fetal-DNA analysis have also been recently reviewed.29,74
Medical Therapy

A decision to initiate regular transfusions in patients with b-thalassemia may be difficult and should be based on the presence and severity of the symptoms and signs of anemia, including failure of growth and development. Only rarely is genotyping helpful in this decision. The goals of transfusion include correction of anemia, suppression of erythropoiesis, and inhibition of increased gastrointestinal absorption of iron. Hypertransfusion and supertransfusion

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15,000

Increased risk of complications Thalassemia major Adequate iron chelation Normal hepatic iron concentration

50

40

10,000
Homozygous hemochromatosis

30

20 5,000
Threshold for cardiac disease and early death Heterozygous hemochromatosis

Optimal iron chelation level

10

0 0 10 20 30 40 50

Age (years)
Figure 4. Hepatic Iron Burden over Time and the Effect of Various Hepatic Iron Concentrations in Patients with Thalassemia Major, Homozygous Hemochromatosis, and Heterozygous Hemochromatosis. Because efforts to maintain normal hepatic iron concentrations in patients with thalassemia major are frequently associated with adverse reactions to deferoxamine, an optimal range within which hepatic iron may be safely maintained, derived in part from clinical experience with the iron-loading disorder hereditary hemochromatosis, has been proposed. In a proportion of patients who are heterozygous for hemochromatosis, moderate iron loading corresponding to concentrations of approximately 3.2 to 7 mg of iron per gram of liver, dry weight (indicated in yellow), is associated with normal survival without complications of iron overload.60 In patients who are homozygous for this disorder, concentrations exceeding this range, up to approximately 15 mg of iron per gram of liver, dry weight (indicated in blue), are associated with an increased risk of complications of iron overload,61 whereas maintenance of levels exceeding 15 mg of iron per gram of liver, dry weight, greatly increases the risk of cardiac disease and early death in patients with thalassemia major.52 In patients with thalassemia major who receive regular transfusions, the rate of iron loading is much more accelerated and death usually occurs before the third decade of life. The goal of treatment is the maintenance of a body-iron burden corresponding to a hepatic iron concentration of approximately 3.2 to 7 mg of iron per gram of liver, dry weight, achievable with regular deferoxamine therapy.40 The serum ferritin concentrations corresponding to these ranges of hepatic iron are not clearly defined.

regimens, which achieve these goals but are associated with substantial iron loading,40,75 have been supplanted by regimens in which the hemoglobin concentration before transfusion does not exceed 9.5 g per deciliter.76 These newer regimens are associated with both adequate marrow suppression and relatively lower rates of iron accumulation. The beneficial effects of iron-chelating therapy with parenteral deferoxamine, the only chelating agent widely available for clinical use, on the complications of iron loading have recently been reviewed.40 As a result of programs of deferoxamine therapy, the prognosis for patients in countries able to afford this therapy has greatly improved, in contrast to the prognosis for patients in developing countries, where widespread implementation of this regimen is still awaited. Adequate deferoxamine therapy prevents early death from cardiac disease: maintenance of body iron burdens corresponding to hepatic iron concentrations of less than 15 mg per gram, dry weight, greatly de-

crease the risk of clinical disease.52 Nearly normal concentrations of hepatic iron can be maintained with modern regimens of deferoxamine. Moreover, deferoxamine arrests the progression of hepatic fibrosis to cirrhosis, even when administered in regimens that stabilize, rather than reduce, the body iron burden.77 The importance of this finding in the seminal study that ushered in the modern era of deferoxamine therapy is highlighted by evidence that in another form of iron overload, hereditary hemochromatosis, progression of hepatic fibrosis is a critical event associated with an increased risk of death.61 A favorable effect of a sustained reduction in body iron is also suggested by the relatively low prevalence of thyroid, parathyroid, and adrenal abnormalities in the modern era.78 In parallel, early and intensive deferoxamine therapy may increase the incidence of normal sexual maturation,78 but it apparently does not reverse established abnormalities.40 Similarly, although deferoxamine prevents diabetes mellitus,52 there is
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no evidence that it can reverse this complication. In summary, modern regimens of subcutaneous deferoxamine may extend survival free of many complications of iron overload, if body iron is reduced or maintained below critical concentrations.40,52,53,79 A balance between the effectiveness of deferoxamine and its toxicity the latter observed primarily in the presence of relatively low body iron burdens80 can be maintained through regular determinations of body iron burden. In clinical practice, the serum ferritin concentration is commonly used to assess the effectiveness of treatment. It is increasingly recognized that reliance on this test may lead to errors in management; changes in body iron account for little more than half the variation in serum ferritin concentrations.81 By contrast, the measurement of hepatic iron stores, whose concentrations are highly correlated with total body iron stores,82 provides the most quantitative, specific, and sensitive method of evaluating iron burden in patients with thalassemia. Determination of hepatic iron concentrations in liver-biopsy specimens obtained with ultrasonographic guidance is safe and permits rational adjustments in iron-chelating therapy.40 Magnetic susceptometry provides a direct measure of hepatic iron stores that is quantitatively equivalent to that determined by biopsy of at least 0.6 mg of liver, dry weight,83 over a range of iron concentrations.84 Magnetic susceptometry is currently available in only two centers worldwide. By contrast, the more widely available technique of magnetic resonance imaging fails to provide accurate quantitation of hepatic iron concentrations in patients with severe iron overload, hepatic fibrosis, or both.85
Bone Marrow Transplantation

Experimental Therapies
Chelators Other Than Deferoxamine

Bone marrow transplantation from HLA-identical donors has been successfully performed worldwide in over 1000 patients with severe b-thalassemia.86 Outcomes after transplantation are greatly influenced by the presence of hepatomegaly, portal fibrosis, and ineffective chelating therapy before transplantation.87 Children without any of these risk factors have rates of survival and disease-free survival exceeding 90 percent three years after transplantation. In those with all three risk factors, and in most adults, the rates are approximately 60 percent. Lower success rates are reported at smaller centers.87 Complications include a rate of chronic graft-versus-host-disease ranging from 2 to 8 percent and a variable incidence of mixed chimerism.86 Post-transplantation management of preexisting hepatic iron overload, iron-induced cardiac dysfunction, and viral hepatitis may prevent progression of these processes.86 There is interest in experimental approaches to bone marrow replacement in patients with thalassemia, including cord-blood transplantation,88 the use of unrelated phenotypically matched donors,89 and in utero transplantation.90
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Difficulties associated with deferoxamine therapy have led to a search for alternatives, including orally active iron chelators. The administration of one agent, deferiprone, was reported to have a favorable short-term effect on body iron in the one study in which serial systematic determinations of hepatic iron were obtained.91 Two subsequent long-term studies have suggested that hepatic iron may stabilize at or increase to concentrations associated with an increased risk of cardiac disease and early death52 in approximately half of patients.92,93 Previously recognized adverse effects of deferiprone included embryotoxicity, teratogenicity, neutropenia, and agranulocytosis.94 Long-term treatment has been reported to be associated with progression of hepatic fibrosis; the odds of progression of fibrosis were estimated to increase by a factor of 5.8 with each additional year of deferiprone therapy.95 In another study, four deaths due to cardiac failure were reported during long-term therapy.92 The results of these clinical trials virtually recapitulate those in two animal species, in which deferiprone and a structurally similar compound were shown to increase hepatic and cardiac iron loading, worsen hepatic fibrosis, and induce cardiac and musculoskeletal fibrosis.95,96 Taken together, these data suggest that deferiprone does not adequately control body iron in a substantial proportion of patients and may promote worsening of hepatic fibrosis. These studies support cautions previously expressed about the long-term administration of this agent.97 The results of long-term follow-up of the effectiveness of other modes of administration of deferoxamine are awaited. These include deferoxamine attached to high-molecular starch,98 administered in twice-daily subcutaneous boluses,99 and given in a lipid vehicle, permitting slow release.100
Augmentation of Fetal-Hemoglobin Synthesis

Several trials have attempted to augment the synthesis of fetal hemoglobin in an effort to ameliorate the severity of b-thalassemia.101 Administration of intravenous 5-azacytidine was associated with increases in the hemoglobin concentration in a few patients102; the potential toxicity of the drug later shifted interest to less toxic alternatives. Therapy with hydroxyurea,103,104 butyric acid compounds,105 and these agents in combination106 has reduced or eliminated transfusion requirements in some patients. Other studies have reported only small increases in fetal and total hemoglobin concentrations during the administration of hydroxyurea107,108 and both intravenous109,110 and oral111,112 butyrate compounds. How can the augmentation of fetal hemoglobin be optimized? Studies in humans and animal models of b-thalassemia, including transgenic mice,113 have

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suggested that increases in the production of g-globin chains may be influenced by the degree of erythroid marrow expansion, sequential administration of specific combinations of agents, the degree to which the g-globin gene is already partially activated, or all of these.113-116 Furthermore, the striking clinical responses observed in patients with mutations104-106 that delete specific sequences within the b-globin gene cluster that may have a key role in the silencing of adjacent genes117 indicate that delineation of some cis sequences may influence the inducibility of the g-globin gene. Although they address a highly desirable, costeffective goal in b-thalassemia, therapies to increase the synthesis of fetal hemoglobin in the disorder have, with few exceptions, proved disappointing to date. Nonetheless, important avenues to be pursued in further studies include the identification of specific mutations that may respond to therapy, particularly with specific combinations of agents.
Gene Therapy

Supported in part by research grants from the Medical Research Council of Canada, the Ontario Heart and Stroke Foundation, and the Ontario Thalassemia Foundation. Dr. Olivieri is the recipient of a Scientist Award from the Medical Research Council of Canada.

I am indebted to David Nathan, David Weatherall, Gary Brittenham, John Porter, Alan Schechter, Elliott Vichinsky, Brenda Gallie, Helen Chan, Peter Durie, John Dick, Marc Giacomelli, Paul Ranalli, Arthur Schafer, Michele Brill-Edwards, Lori West, Miriam Kaufman, Michael Langlois, David Kern, Bob Phillips, Maria Muraca, Bill Graham, Rhonda Love, Doug Templeton, John Polanyi, and Michael Baker, without whose support this review would not have been written.

REFERENCES
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Permanent correction of genetic deficit of the hematopoietic system requires the transfer of genes into stem cells and long-term, high-level, lineagespecific expression of these cells after autologous transplantation; mature cells and committed progenitors do not have the proliferative capacity to reconstitute the entire hematopoietic system. Over the past decade, there has been progress in the development of transduction methods and vectors.118 Remaining problems include the identification of all sequences required for stable, high-level expression of the genes and the development of more effective and safe vectors for the transfer of genes.119 Another approach, correction of the defective gene by site-directed recombination, is feasible, but current methods lack the degree of efficiency required.120
CONCLUSIONS

Among the first diseases to be studied at the molecular level, the b-thalassemias remain a model for understanding the relation between the molecular pathology of a disease and its clinical diversity. At the same time, these disorders have become an increasingly important part of clinical practice in all countries with large populations from the tropics. The marked increase in survival, to the fifth decade of life, of patients with well-managed b-thalassemia in developed countries represents one of the most dramatic alterations in morbidity and mortality associated with a genetic disease in this century. Still, nearly 75 years after the fascinating initial description of peculiar bone changes and other signs and symptoms of the disorder, the b-thalassemias have emerged as a huge public health problem worldwide. They remain a therapeutic challenge for the next millennium.

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