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Introduction

The thalassemias, a heterogeneous family of inherited dis-orders of hemoglobin synthesis, were first recognized independently in the USA and Italy in the years between 1925 and 1927.1 The word thalassemia owes its name to anattempt, mistaken as it turned out later, to relate the diseasesto Mediterranean populations; thalassa is from the Greekword for sea.

It is now apparent that the thalassemias are the worldscommonest monogenic diseases and are widespread amongraces ranging from the Mediterranean region, through theMiddle East and Indian subcontinent, to Southeast Asia.Many countries in these regions have, over the last 30 years,gone through a remarkable demographic change in the pattern of their illnesses. With improvements in hygiene andpublic health measures, very high infant and childhood mor-talities due to infection and malnutrition have fallen. In thepast, babies born with serious genetic blood diseases wouldhave been unlikely to survive the first years of life, but thescene has now changed dramatically. As these countriesundergo this demographic transition, the majority of thesechildren are now surviving long enough to come to diagnosisand to require management. And because the symptomatictreatment of thalassemia is expensive, this change in the pat-tern of childhood illness will place an increasing drain on theresources of countries in which the disease occurs at a highfrequency.2

These diseases are also assuming an increasing importancein the clinical practice of pediatricians in the richer countries.

Genetics and classification

Genetic control of hemoglobin synthesis

A great deal is known about the structure, genetic regulation

and synthesis of hemoglobin. Only those aspects of particularimportance for an understanding of the thalassemias aresummarized here. Readers are referred to more extensivereviews and monographs.36

Different hemoglobins, each adapted to the particular oxy-gen requirements at each stage of development, are synthes-ized in the embryo, fetus and adult. All hemoglobins have asimilar tetrameric structure, consisting of two different pairsof globin chains, each attached to a heme moiety. Adult andfetal hemoglobins have chains combined with chains(HbA, 22), chains (HbA2, 22) and chains (HbF, 22). In embryonic life, -like chains called chains combine with chains to produce Hb Portland (22), or with chains toform Hb Gower 1 (22), and and chains combine to formHb Gower 2 (22). Fetal hemoglobin is itself heterogeneous;there are two kinds of chains, which differ in their aminoacid compositions only at position 136, where they haveeither glycine or alanine. Those chains with glycine arecalled G chains and those with alanine A chains. The G andA chains are the products of separate (G and A) loci.

The different globin chains are controlled by two mainfamilies of globin genes (Fig. 13.1). The -like globin genes are arranged in a linked cluster on chromosome 11, which is distributed over approximately 60 kb (kilobase or 1000nucleotide bases). They are arranged in the order 5 to 3 (leftto right) -G-A---. The symbol is used to described apseudogene, probably a burnt-out evolutionary remnant of a once-active gene. The -like globin genes also form a clus-ter, in this case on chromosome 16. They are distributed in theorder 5---1-2-1-3.

In order to appreciate the molecular basis for the thalass-emias, it is important to understand, at least in outline, something of the structure of the globin genes, how they are regulated, and how their products are synthesized andunite with heme to form hemoglobin molecules in the red cellprecursors.4,6

Each globin gene consists of a string of nucleotide basesthat are divided into coding sequences, called exons, and

ThalassemiasNancy F. Olivieri and David J. Weatherall13

Pediatric Hematology, Third Edition Edited By Robert J. Arceci, Ian M. Hann, Owen P. Smith

Copyright 2006 by Blackwell Publishing Ltd

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noncoding regions known as intervening sequences (IVS) orintrons. In the 5 (left-hand) noncoding or flanking regions ofglobin genes, there are blocks of nucleotide homology, whichare found in similar positions in many species. Three suchregions, called promoter elements, play a major role in thetranscription of the structural genes. The globin gene clusterscontain other elements that play an important part in promot-ing erythroid-specific gene expression and in coordinatingthe changes in globin gene activity at different stages duringdevelopment. These include enhancers, i.e., sequences thatincrease gene expression despite being located at a variabledistance from a particular gene, and master regulatorysequences, called the locus control region (LCR) in the case ofthe -globin gene family and HS40 in the case of the -genecomplex, which lie upstream from the globin gene clustersand are responsible for their activation in erythroid tissue.Each of these regulatory sequences has a modular structurecomprising an array of short nucleotide motifs that representbinding sites for transcriptional activators or repressors,molecules involved in the activation or repression of globingene production in different cell types and at different stagesof development.

Each of these regulatory regions binds a number of erythroid-specific factors, including GATA-1 and NF-E2,thereby activating the LCR, which renders the entire -globingene cluster transcriptionally active. It seems likely that the LCR and HS40 regions come into apposition with the promoter regions of each of the globin genes in turn and,together with a complex collection of transcription factorsand other proteins, form an initiation complex so that indi-vidual genes are transcribed.

When a globin gene is transcribed, messenger RNA(mRNA) is synthesized from one of its strands by the actionof RNA polymerase. The primary transcription product is a large mRNA precursor, which contains both intron andexon sequences (Fig. 13.2). While in the nucleus this moleculeundergoes a remarkable series of modifications; the introns

are removed and the exons are spliced together. This is a multistep process that requires certain structural features ofthe mRNA precursor, notably the nucleotides GT at the 5end and AG at the 3 end of intronexon junctions. We will discuss the importance of these sequences when we considerthe mutations that cause thalassemia. The mRNAs are nowmodified at both their 5 and 3 ends and move into the cyto-plasm of the red cell precursor to act as a template for globinchain production.

Amino acids are transported to the mRNA template on carrier molecules called transfer RNAs (tRNAs). There arespecific tRNAs for each amino acid. The order of amino acidsin a globin chain is determined by a triplet code, where threebases (codons) code for a particular amino acid. The tRNAsalso contain three bases, or anticodons, that are complement-ary to the mRNA codons for particular amino acids. Hencethe tRNAs carry amino acids to the template, find the rightposition by codon/anticodon base pairing, and initiateglobin chain synthesis. When the first tRNA is in position, acomplex is formed between several protein initiation factorsand the subunits of the ribosome that is to hold the growingpeptide chains together on the mRNA as it is translated. A second tRNA moves in alongside and the two amino acidsare united by a peptide bond; the globin chain is now twoamino acid residues long. This process is continued as themessage is translated, from left to right, until a specific codonfor termination is reached, whereupon the finished globinchain drops off the ribosome mRNA complex and the ribo-somal subunits are recycled. The finished globin chain com-bines with heme and three of its fellows to form a definitivehemoglobin molecule.

The developmental switches from embryonic to fetal and fetal to adult hemoglobin production are synchronizedthroughout the different organs of hemopoiesis that functionat various times of development.4,6 The way in which theseswitches are regulated is not yet completely understood. It isbelieved that the LCR becomes spatially related sequentially

31 31 104 105

1 kb

16 11

Embryo Fetus Adult

32 99 100

1 G A 1212

22Hb Gower 1

22Hb Portland

22Hb Gower 2

22HbF

22HbA

22HbA2

30

Fig. 13.1 Genetic control of human hemoglobin synthesis. The - and -globin gene clusters on chromosomes 16 and 11 are shown, together with thedifferent hemoglobins produced in embryonic, fetal and adult life. In the extended representations of the 1- and -globin genes the exons are shown in dark shading, the introns unshaded, and the 5 and 3 noncoding regions are shown in light shading.

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to the , and finally and chains at different times duringfetal development. Why this happens is not clear, although it is possible that there are specific DNA-binding proteinsinvolved in the activation or repression of these genes at dif-ferent developmental stages.

Classification

The thalassemias can be defined as a heterogeneous group of genetic disorders of hemoglobin synthesis, all of whichresult from a reduced rate of production of one or more of the globin chains of hemoglobin. This basic defect results inimbalanced globin chain synthesis, which is the hallmark ofall forms of thalassemia.1

The thalassemias can be classified at different levels. Clin-ically, it is useful to divide them into three groups: the severetransfusion-dependent (major) varieties; the symptomlesscarrier states (minor) varieties; and a group of conditions of intermediate severity that fall under the loose headingthalassemia intermedia. This classification is retained be-cause it has implications for both diagnosis and management.

Thalassemias can also be classified at the genetic level intothe , , or thalassemias, according to which globinchain is produced in reduced amounts (Table 13.1). In somethalassemias, no globin chain is synthesized at all, and hencethey are called 0 or 0 thalassemias, whereas in others

Table 13.1 Classification of the common thalassemias and relateddisorders.

Thalassemia+, 0

Thalassemia()+ Hb Lepore thalassemia()0

(A)0

Thalassemia()0

Thalassemia

or thalassemia associated with -chain variantsHbS thalassemiaHbE thalassemiaMany others

Thalassemia+ (deletion)+ (nondeletion)0

Hereditary persistence of HbF

Deletion ()0

Nondeletion A+, G+

Unlinked to -globin gene cluster

Excision of intronsSplicing of exons

mRNA precursor

Gene

Translation

Nucleus

Cytoplasm

FlankingIVS 2IVS 1Flanking

CACCC

CCAAT

TATA

ATG

NC GT AG GT AG NC

TAA

ATAAA

3''5

AAAAn

AAAAn

5' CAP

AAAAnUAA

AAUUGA CGU UUC

GGG GGGRibosome

Transfer RNA

Amino acid

Growing chain

Processedchain

Finishedchain

Processed mRNA

Fig. 13.2 A schematic representation of gene action and proteinsynthesis. Reproduced from Weatherall DJ, Clegg JB, eds. The

Thalassaemia Syndromes, Blackwell Science Ltd, 2001.

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some globin chain is produced but at a reduced rate; these are designated + or + thalassemias. The thalassemias, inwhich there is defective and chain synthesis, can be subdivided in the same way, i.e., into ()+ and ()0 varieties.

Because the thalassemias occur in populations in whichstructural hemoglobin variants are also common, it is notunusual to inherit a thalassemia gene from one parent and a gene for a structural variant from the other. Furthermore,since both and thalassemia occur commonly in somecountries, individuals may receive genes for both types. Allthese different interactions produce an extremely complexand clinically diverse family of genetic disorders, which rangein severity from death in utero to extremely mild, symptom-less, hypochromic anemias.

Despite their genetic complexity, most thalassemias areinherited in a mendelian recessive or codominant fashion.Heterozygotes are usually symptomless, while more severelyaffected patients are either homozygous for or tha-lassemia, or compound heterozygous for different molecularforms of the diseases.

Distribution

A world map of the distribution of the thalassemias is shown

in Figs 13.3 and 13.4. Several detailed accounts of their fre-quency and population genetics have been reported.1,7

The 0 thalassemias are found predominantly in SoutheastAsia and in the Mediterranean islands. The + thalassemiasoccur widely throughout Africa, the Mediterranean region,the Middle East, parts of the Indian subcontinent, andthroughout Southeast Asia. They occur at remarkably highfrequencies in some populations, achieving carrier rates ofbetween 40 and 80%.

The thalassemias have a distribution similar to that of the thalassemias. With the exception of a few countries, the thalassemias are less common in Africa, extremely frequentin some of the Mediterranean island populations, and occurat variable frequencies throughout the Middle East, theIndian subcontinent and parts of Southeast Asia. As we shallsee later, the structural hemoglobin variant, HbE, is asso-ciated with the phenotype of a mild form of thalassemia.This also reaches extremely high gene frequencies in easternparts of India, Myanmar, and in many countries in SoutheastAsia. Thus the interaction of HbE and thalassemia, HbE thalassemia, is the most important form of the disease inthese regions.

There is increasing evidence that these high gene fre-quencies for the different forms of thalassemia have beenmaintained by heterozygote advantage against severe forms

IVS 1 - 110 GAIVS 1 - 5 GCIVS 1 - 6 TCCODON 39 CAGTAGCODON 8 2bp DEL

CODON 6 - 1bpIVS 1 - 1GAIVS 2 - 1 GAIVS 2 - 745 CGCODON 39 CAGTAGIVS 1 - 6TCIVS 1 - 110 GA

IVS 1 - 5 GCIVS 1 - 1 GTCODONS 41 - 42.4bp DEL.CODONS 26 GAGAAG(HbE)

IVS 1 - 5 GC619 bp DELETIONCODON 8/9 + GIVS 1 - 1 GTCODONS 41 - 42.4bp DEL.

IVS 1 - 5 GC

IVS 2 - 654 CTCODONS 41 - 42.4bp DEL.CODON 17 AAGTAGCODON 26 GAGAAG(HbE)-28 AG-29 AG

-29 AG-88 CTCODON 24 TAPOLY-A TC

Fig. 13.3 World distribution of the different mutations that cause thalassemia. IVS, intervening sequence.

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of malaria, predominantly Plasmodium falciparum. One of themost remarkable features of the world distribution of thesediseases is that in each high-frequency population there aredifferent sets of mutations (Figs 13.3 and 13.4). This tells usthat these diseases have arisen by new mutations and thenbeen expanded very rapidly due to local selection by malaria.The fact that the mutations are so different among popula-tions indicates that this selective force is, in evolutionary terms,quite recent, probably not more than a few thousand years.

Pathophysiology

To appreciate the pathophysiology of the thalassemias, it isnecessary to understand both their molecular pathology andhow globin chain imbalance causes the characteristic ineffect-ive erythropoiesis and shortened red cell survival.

Molecular pathology

Although the basic principles are similar, the patterns of the mutations that cause and thalassemia are different.Because of their greater clinical importance, we consider the thalassemias first.

Over 150 different mutations have been described in patientswith thalassemia (Fig. 13.5).8,9 Unlike the thalassemias,major deletions causing thalassemia are unusual. The bulkof -thalassemia mutations are single base changes or smalldeletions or insertions of one or two bases at critical pointsalong the genes. Remarkably, they occur in both introns andexons, and outside the coding regions of the genes.

Some substitutions, called nonsense mutations, result in a single base change in an exon that generates a stop codon in the coding region of the mRNA. This, of course, causes premature termination of globin chain synthesis and leads tothe production of a shortened and nonviable -globin chain.Other exon mutations cause frameshifts, which result in oneor more bases being lost or inserted so that the reading frameof the genetic code is thrown out of phase or a new stop codonis produced. Mutations within introns or exons, or at theirjunctions, may interfere with the mechanism of splicing theexons together after the introns have been removed duringprocessing of the mRNA precursor. For example, single substitutions at the invariant GT or AG sequences at theintron/exon junctions prevent splicing altogether and cause0 thalassemia. The sequences adjacent to the GT and AGsequences are highly conserved and are also involved insplicing; several -thalassemia mutations involve this region

-- MED- 3.7 I

-- SEA- 3.7 I

- 4.2- 3.7 IIIT

1-15%

60%(- 3.7)(T )

40 - 80%(- 3.7 I)

5 - 40%(- 3.7 I)

5 - 80%

5 - 15%

Thalassemia

Thalassemia

+

0

Fig. 13.4 World distribution of the different varieties of thalassemia. The three different forms of + thalassemia due to deletions of a single -globin geneinvolving the loss of 3.7 kb of the -gene cluster are represented as types I, II and III. The common deletional forms of 0 thalassemia are designated MED(Mediterranean) and SEA (Southeast Asian). The percentages indicate the approximate frequency of these different genes.

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and are associated with variable degrees of defective -globinproduction. Mutations in sequences in exons that resembleconsensus sequences at the intron/exon junctions may activ-ate cryptic splice sites. For example, there is a sequence thatresembles the IVS-1 consensus site and spans codons 2427 ofexon 1 of the -globin gene; mutations at codons 19 (AG),26 (GA), and 27 (GT) result in both a reduced amount ofmRNA due to abnormal splicing and an amino acid substitu-tion encoded by the mRNA that is spliced normally andtranslated into protein. The abnormal hemoglobins producedare hemoglobins Malay, E and Knossos, each of which isassociated with a mild -thalassemia phenotype.

Single base substitutions are also found in the flankingregions of the -globin genes. Those which involve the pro-moter elements downregulate -globin gene transcription,and are usually associated with a mild form of thalassemia.Other mutations involving the 3 end of the -globin mRNAinterfere with its processing and produce severe -thalassemiaphenotypes.

Because there are so many different -thalassemia mutations,it follows that many patients who are apparently homozy-gous for the disease are, in fact, compound heterozygotes fortwo different molecular lesions. Rarely, patients are encoun-tered with forms of thalassemia in which the HbA2 level,which is usually raised in carriers, is normal. Usually thisresults from the coinheritance of and thalassemia.

The thalassemias are also divided into the ()+ and ()0 forms. The ()+ thalassemias result from misalign-ment of the and globin genes during meiosis with the production of fusion genes. These give rise to structuralhemoglobin variants called the Lepore hemoglobins, after thefamily name of the first patient to be identified with this con-dition. Because the genes that direct the fusion chains have-globin gene promoter regions that contain mutations whichresult in their ineffective transcription, the chains are synthesized at a reduced rate and hence are associated withthe phenotype of thalassemia. The different forms of ()0

thalassemia all result from long deletions of the -globin genecluster that remove the and genes, and leave either one orboth the -globin genes intact. Longer deletions that removethe -globin LCR and all or most of the cluster completelyinactivate the gene complex and result in ()0 thalassemia.9,10

The molecular pathology and genetics of the thalasse-mias are more complicated than that of thalassemia, largelybecause there are two functional -globin genes on each pairof chromosomes.9,11,12 The normal -globin genotype can be written /. The 0 thalassemias result from a family ofdifferent-sized deletions that remove both -globin genes;the homozygous and heterozygous states are designated / and /, respectively. Rarely, 0 thalassemia mayresult from deletions involving a region similar to the -globin LCR, 40 kb upstream from the -globin gene cluster,or from short truncations of the end of the short arm of chromosome 16.

The molecular basis for the + thalassemias is more compli-cated. In some cases they result from deletions that removeone of the linked pairs of -globin genes, /, leaving theother intact, while in others both -globin genes are intact butone of them has a mutation that either partially or completelyinactivates it, T/.

The deletion forms of + thalassemia are further classifiedinto the particular size of the underlying deletion. There aretwo common varieties, involving loss of either 3.7 or 4.2 kb ofDNA; they are designated 3.7 and 4.2 respectively. It turnsout that the former is quite heterogeneous, depending on thesite of the abnormal genetic cross-over event that underliesthe deletion. These deletions are thought to be due to mis-alignment and reciprocal cross-over between the -globingene segments at meiosis; this mechanism results in one chromosome with a single ( ) gene and the opposite of thepair with a triplicated () -gene arrangement.

Nondeletional forms of thalassemia, in which the -globin genes are intact, are caused by mutations that are verysimilar to those that cause thalassemia. Some result from

Deletions

Point mutations

SPSP FSNS

FSNS

I

IVS 1

CP FSNS

FSNS

SP SP CL

100 bp

IVS 21 32

Fig. 13.5 Different classes of mutations of the -globin gene involved in thalassemia. FS, frameshift; NS, nonsense; SP, splicing; P, promoter; CL, polyA addition site mutations; IVS, intervening sequence.

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initiation or splice mutations, or the production of a highlyunstable globin that is incapable of producing a viabletetramer. Another particularly common form, found inSoutheast Asia, results from a single base change in the termination codon UAA, which changes to CAA. The latter is the code for the amino acid glutamine. Hence, when theribosomes reach this point, instead of the chain terminating,mRNA which is not normally transcribed is read throughuntil another stop codon is reached. Thus, an elongated -globin chain is produced that is synthesized at a reduced rate;the resulting variant is called Hb Constant Spring after thename of the town in Jamaica where it was first discovered. Itoccurs in about 25% of the population of Thailand and otherregions of Southeast Asia. Because the termination codon canchange to yield several different codons, this variant is onlyone of a family of chain-termination mutants. Another com-mon form of nondeletional thalassemia, which is found in the Middle East, results from a single base change in thehighly conserved sequence of the 3 coding region of the -globin gene, AATAAA, which is changed to AATAAG. Thisis the signal site for polyadenylation of globin mRNA, a pro-cess that appears to stabilize its passage into the cytoplasm.This mutation results in marked reduction in -globin chainproduction from the affected locus.

In addition to these common forms of thalassemia, there is a syndrome characterized by mild thalassemia andmental retardation (ATR) that is being recognized increas-ingly in many different populations. By combining clinicaland molecular studies, it has been possible to subclassify this condition into two main syndromes, one encoded onchromosome 16 (ATR-16) and another on the X chromosome(ATR-X). ATR-16 is associated with relatively mild mentalretardation and results from a variety of long deletions thatremove the end of the short arm of chromosome 16. Thesemay occur alone or as part of a chromosomal translocation.10

ATR-X, which is characterized by a more severe form of men-tal retardation with a severe dysmorphologic picture, resultsfrom mutations of a gene on the X chromosome identified asATRX.11 The gene product is a DNA helicase that appears tobe one of a family of trans-acting proteins involved with generegulation through the remodeling of chromatin.12 There isalso a form of thalassemia associated with myelodysplasia,particularly in elderly patients.13 Recently it has been foundthat this condition, like ATR, is also associated with acquiredsomatic mutations in ATRX.14

Cellular pathology

Although the basic defect, imbalanced globin chain synthesis,is similar in all types of thalassemia, the consequences ofexcess or chain production in the and thalassemias are quite different.1 Excess chains that are produced in thalassemia are unable to form a hemoglobin tetramer andprecipitate in the red cell precursors. On the other hand, the

excess of and chains produced at different developmentalstages in the thalassemias are able to form homotetramerswhich, although unstable, are viable and form soluble hemo-globin molecules called Hb Barts (4) and HbH (4). It is thesefundamental differences in the behavior of the excess chainsin the two common classes of thalassemia that are responsiblefor the major differences in their cellular pathology.

The -thalassemias

The excess of chains produced in thalassemia is highlyunstable, and rapidly precipitates and becomes associatedwith the membrane of red cell precursors and red cells. Thisphenomenon leads to extensive intramedullary destructionof red cell precursors, probably through a variety of complexmechanisms including interference with cell division andoxidative damage to the precursor membranes.1518 Becausethey contain large inclusion bodies, such red cells as do reachthe peripheral blood are damaged in their passage throughthe spleen and their membranes also suffer severe oxidativeinjury due to the action of heme liberated from denaturedhemoglobin and the excess of iron that accumulates in thethalassemic red cell. Thus the anemia of thalassemia reflectsa combination of ineffective erythropoiesis combined withreduced red cell survival.

Small populations of red cell precursors retain the capacityfor producing chains of HbF in extrauterine life. In tha-lassemics these cells come under intense selection; the excessof chains is smaller because some of them combine with chains to produce HbF. Thus, the baseline level of fetalhemoglobin is elevated in thalassemia. Cell selection occursthroughout the lifespan of the HbF-rich population;1 it is alsoapparent that there are a number of genetic factors that modify the ability to make HbF in response to severe anemia.19

These factors combine to produce increased levels of HbF in all forms of severe thalassemia. Since -chain synthesis is unaffected in thalassemia, heterozygotes usually have anelevated level of HbA2, another important diagnostic feature.

The profound anemia of thalassemia, and the productionof red cell populations rich in HbF and a high oxygen affinity,combine to cause severe hypoxia and stimulate erythropoi-etin production. This, in turn, leads to extensive expansion ofthe ineffective erythroid mass with consequent bone changes,increased iron absorption, a high metabolic rate, and many of the other clinical features of severe thalassemia. Thebombardment of the spleen with abnormal red cells causesincreasing splenomegaly; hence the disease may be com-plicated by trapping of part of the circulating red cell mass in the spleen which, together with sequestration of white cells and platelets, may produce the classical picture of severehypersplenism.

Many of these features can be reversed by suppressingineffective erythropoiesis by transfusion, which leads in turnto increased iron overload. The resulting pathology can

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be best appreciated against the background of normal ironmetabolism.20 In normal individuals, tight binding of plasmairon to the transport protein transferrin prevents the catalyticactivity of iron in free-radical production.21 In heavily iron-loaded patients, transferrin becomes fully saturated and anontransferrin-bound fraction of iron becomes detectable inplasma. This may accelerate the formation of free hydroxylradicals and results in accelerated iron loading in tissues,with consequent organ damage and dysfunction. In patientswith iron overload, excess iron is deposited in both reticulo-endothelial cells (where it is relatively harmless) and paren-chymal tissue, primarily myocytes and hepatocytes (where it may cause significant damage). The toxicity of iron is mediated, in part, by its catalysis of reactions that gener-ate free hydroxyl radicals, propagators of oxygen-relateddamage.21

Clearly, therefore, it is possible to relate most of the clinicalfeatures of severe thalassemia to the consequences of defect-ive -globin production, the deleterious results of excess -

globin chain synthesis on erythroid maturation and survival,and the effects of iron loading resulting from increasedabsorption and blood transfusion (Fig. 13.6). If these prin-ciples are appreciated, it is easy to understand why someforms of thalassemia are associated with a much milderphenotype. Indeed, all the known factors that modify thephenotype of this disease act by reducing the amount ofglobin chain imbalance. They include the coinheritance of thalassemia, the presence of a mild -thalassemia allele, or the cosegregation of a gene that results in a higher thanusual output of fetal hemoglobin.19

The -thalassemias

The cellular pathology of thalassemia, because of the prop-erties of HbH and Hb Barts, is different in many ways to thatof thalassemia.1,11,18 As the result of the generation of thesesoluble tetramers there is less ineffective erythropoiesis.Particularly in the case of HbH, the tetramer tends to pre-

Selective survival ofHbF-containing cells

Hemolysis Destruction ofred blood cell

precursors

Ineffectiveerythropoiesis

Splenomegaly(pooling, plasma

volume expansion)

Excess

22Hbf

Precipitation

High oxygenaffinity of red cells

Anemia

Tissue hypoxia

Erythropoietin Transfusion

Marrow expansion

Increased ironabsorption

Bone deformityIncreased metabolic rate

WastingGout

Folate deficiency

Iron loading

Endocrine deficienciesCirrhosis

Cardiac failureDeath Fig. 13.6 Pathophysiology of thalassemia.

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cipitate as cells age, with the production of inclusion bodies, and hence a major hemolytic component is a feature of thedisorder. The clinical effects of the anemia are exacerbated by the fact that both HbH and Hb Barts are homotetramersand therefore cannot undergo the allosteric changes requiredfor normal oxygen delivery. They behave, in effect, like myo-globin and are unable to give up oxygen at physiologic ten-sions. Hence, high levels of Hb Barts or HbH are associatedwith severe hypoxia.

The pathophysiology of the thalassemias can be bestunderstood in terms of simple gene dosage effects.11 In thehomozygous state for 0 thalassemia ( / ) no chains areproduced. Affected infants have very high levels of Hb Bartswith some embryonic hemoglobin. Although their hemo-globin concentration might be compatible with intrauterinelife if the structure of their hemoglobin were normal, the factthat it is nearly all Hb Barts renders them seriously hypoxic.Most affected infants are stillborn with all the signs of grossintrauterine hypoxia. The compound heterozygous state for0 and + thalassemia ( / ) results in less chain imbalanceand is compatible with survival, a condition called HbH dis-ease. This disorder is characterized by a variable hemolyticanemia; adaptation to the level of anemia is often unsatis-factory because HbH, like Hb Barts, is unable to function asan oxygen carrier.

The heterozygous state for 0 thalassemia ( /) and thehomozygous state for the deletion form of + thalassemia ( / ) are associated with a mild hypochromic anemia,very similar to -thalassemia trait. Although a few red cellscontain HbH inclusions in 0-thalassemia trait, they are notobserved in +-thalassemia trait, suggesting that there mustbe a critical level of excess chains required to form viable 4 tetramers. Interestingly, the homozygous states for thenondeletion forms of thalassemia (T/T) are associatedwith a more severe deficiency of chains and the clinical phenotype of HbH disease.

Clinical features

The thalassemias

Most children with the severe forms of homozygous or compound heterozygous thalassemia present within thefirst year of life, with failure to thrive, poor feeding, inter-mittent bouts of infection, and general malaise. These infants are pale and, in many cases, splenomegaly is already present.At this stage there are no other specific clinical signs and thediagnosis rests on the hematologic changes, outlined later. Ifthe infant receives regular red cell transfusions, subsequentdevelopment is usually normal and further symptoms do notoccur until puberty, when, if they have not received adequatechelation therapy, the signs of iron loading start to appear. If, on the other hand, the infant is not adequately transfused,

the typical clinical picture of thalassemia major develops. It follows therefore that clinical manifestations of the severeforms of thalassemia can be described in two contexts: in the well-transfused child, and in the child with chronicanemia throughout early childhood.1,22

In the adequately transfused child, early growth and devel-opment are normal and splenomegaly is minimal or absent.If chelation therapy is effective, these children may enter normal puberty and continue to grow and develop normallyinto early adult life.23,24

On the other hand, if chelation therapy is inadequate, thereis a gradual accumulation of iron, the effects of which start to become manifest by the end of the first decade. The normaladolescent growth spurt fails to occur and the hepatic, endocrine and cardiac complications of iron overloading give rise to a variety of symptoms, including diabetes, hyperthyroidism, hypoparathyroidism and progressive liver failure. Secondary sexual development is delayed orabsent.

The commonest cause of death in iron-loaded children,which usually occurs toward the end of the second decade orearly in the third decade, is iron-induced cardiac dysfunc-tion; these patients die either in protracted cardiac failure orsuddenly due to an acute arrhythmia, often precipitated by infection.

The clinical picture in inadequately transfused patients isquite different.1 The rates of growth and development areretarded and progressive splenomegaly may cause a worsen-ing of the anemia, and is sometimes associated with thrombo-cytopenia. There may be extensive bone marrow expansionleading to deformities of the skull, with marked bossing and overgrowth of the zygomata giving rise to the classicalmongoloid appearance. These bone changes are associatedwith a characteristic radiologic picture that includes a lacytrabecular pattern of the long bones and phalanges, and a char-acteristic hair on end appearance of the skull. These childrenare prone to infection, which may cause a catastrophic dropin the hemoglobin level. Because of the massive expansion ofthe ineffective erythroid mass, they are hypermetabolic, runintermittent fevers, and fail to thrive. They have increasedrequirements for folic acid; deficiency is often associated with worsening of anemia. Because of the increased turnover of red cell precursors, hyperuricemia and secondary goutoccur occasionally. There is also a bleeding tendency, which,although partly explained on the basis of thrombocytopenia,may also be exacerbated by liver damage associated with iron loading, viral hepatitis, or extramedullary hemopoiesis. If these children survive to puberty, they often develop thesame complications of iron loading as well-transfusedpatients; in this case some of the iron accumulation resultsfrom an increased rate of gastrointestinal absorption.

The prognosis for inadequately transfused thalassemicchildren is poor. If they receive no transfusions, they may diewithin the first 2 years; if maintained at a low hemoglobin

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level throughout childhood, they usually die of infection orother intercurrent illness in early childhood. If they survive toreach puberty, they succumb to the effects of iron accumula-tion in the same way as the adequately transfused but poorlychelated child.

It should be emphasized that poor growth in children with thalassemia is not restricted to those who are inadequatelytransfused or iron chelated. This problem is also observed in well-managed patients for reasons that are not entirelyunderstood, and may include iron-induced selective centralhypogonadism, impaired growth hormone responses togrowth hormone-releasing hormone, delay in pubertaldevelopment, zinc deficiency, and over-intensive deferoxam-ine administration.

On presentation, hemoglobin values in the thalassemicchild range from 2 to 8 g/dL. The red cells show markedhypochromia and variation in shape and size; there are manyhypochromic macrocytes and misshapen microcytes, some of which are mere fragments of cells. There is moderatebasophilic stippling and nucleated red cells are always present in the peripheral blood. After splenectomy these may appear in large numbers. The reticulocyte count is onlymoderately elevated. The white cell and platelet counts arenormal unless hypersplenism is present. The bone marrowshows marked erythroid hyperplasia and many of the redcell precursors contain ragged inclusions, best demonstratedby staining with methyl violet, that represent -globin precipitates.

The HbF level is always elevated and it is heterogeneouslydistributed among the red cells. In 0 thalassemia there is no HbA; the hemoglobin consists of F and A2 only. In +

thalassemia the level of HbF ranges from 20 to over 90%. The HbA2 value is usually normal and of no diagnostic value.In vitro globin synthesis studies, involving the labeling of theglobin chains with radioactive amino acids, reveals a markeddegree of globin chain imbalance with an excess of overnon- chain production.

-Thalassemia trait

This is almost invariably asymptomatic and is characterizedby mild anemia; splenomegaly is unusual. There is a slightlyreduced hemoglobin level and a marked reduction in meancell hemoglobin (MCH) and mean cell volume (MCV). Theblood film shows hypochromia, microcytosis and variablebasophilic stippling. In the majority of cases the HbA2 level iselevated to about twice normal (i.e., in the 46% range),while there is a slight elevation of HbF in about 50% of cases. In some populations, notably those of the Mediterranean, -thalassemia trait may be associated with a normal HbA2level. By far the commonest cause is the coinheritance of agene for thalassemia. For genetic counseling (see later), it isvital to distinguish this condition from the different forms of-thalassemia trait.

Intermediate forms of thalassemia

Not all forms of homozygous or compound heterozygous thalassemia are transfusion dependent from early life. Theterm thalassemia intermedia is used to describe a widespectrum of conditions, ranging from those that are almost assevere as thalassemia, with marked anemia and growth re-tardation, to those which are almost as mild as -thalassemiatrait and which may only be discovered on routine hemato-logic examination.19 In the more severe varieties there is obvi-ous growth retardation, bone deformity and failure to thrivefrom early life and, except for a later presentation, the con-dition differs little from the transfusion-dependent forms of the illness. For its management this type of thalassemia inter-media should be considered to be in the same category as thesevere transfusion-dependent form. On the other hand, manyvarieties are associated with good early growth and develop-ment, a satisfactory steady-state hemoglobin level, and mildto moderate splenomegaly. Even in these patients, several important complications may develop as these patients growolder, including increasing bone deformity, progressive osteo-porosis with spontaneous fractures, leg ulcers, folate defi-ciency, hypersplenism, progressive anemia, and the effects ofsystemic iron overload due to increased intestinal absorption.

Thalassemia associated with -globin structuralvariants

Although thalassemia has been found in association withmany different -globin chain variants, the only common disorders of this type are due to the coinheritance of HbS,HbC, or HbE.1,3

HbS thalassemiaHbS thalassemia varies considerably in its clinical mani-festations, depending mainly on the nature of the associated -thalassemia gene. HbS 0 thalassemia, in which no HbA isproduced, is often indistinguishable from sickle cell anemia.Similarly, HbS + thalassemia in which the thalassemia generesults in a very low output of normal chains, and hence alevel of HbA in the 510% range, often runs a severe course.On the other hand, HbS + thalassemia in which the -thalassemia allele is of the mild variety, particularly thoseforms seen in Black populations and in which levels of HbAare in the 3040% range, may be extremely mild and are oftenasymptomatic. The clinical manifestations of the sickling disorders are described in Chapter 10.

HbC thalassemiaHbC thalassemia occurs in West Africa and in theMediterranean population and is characterized by a mild tomoderate form of thalassemia intermedia with the typicalhematologic changes of thalassemia associated with the presence of nearly 100% of target cells in the peripheral blood.

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291

HbE thalassemiaHbE thalassemia is a condition of major importance in eastern parts of India, Bangladesh, Myanmar, and throughoutSoutheast Asia. Since HbE behaves like a mild -thalassemiaallele, it is not surprising that his condition can behave likehomozygous thalassemia. However, what is difficult toexplain is the remarkable clinical variability in its course. Theclinical picture and complications may range from those oftransfusion-dependent homozygous thalassemia (Fig. 13.7)through the milder forms of thalassemia intermedia, as described above. The pattern of complications of the moresevere forms of HbE thalassemia are similar to thosedescribed earlier for thalassemia major; the milder formsbehave like -thalassemia intermedia. The hemoglobin pat-tern depends on the nature of the associated -thalassemiagene; in HbE 0 thalassemia the globin is made up of F and E,whereas in HbE + thalassemia there are variable amounts of HbA.

Thalassemia

The common forms of ()+ thalassemia are the Hb Leporedisorders.1,19 The homozygous state is usually characterizedby a clinical disorder that is indistinguishable from

thalassemia major, although some cases run a milder course. The hemoglobin consists mainly of HbF, with up to 20% HbLepore. Heterozygotes for the different Hb Lepore disordershave the hematologic findings of thalassemia trait, with ahemoglobin pattern that consists of approximately 515% HbLepore and low or normal levels of HbA2.

There are many different molecular varieties of ()0

thalassemia. The homozygous states are characterized by a mild to moderate form of thalassemia intermedia with typical thalassemic red cell changes and a hemoglobin pattern characterized by 100% HbF. Heterozygotes have mild thalassemic red cell changes, with levels of HbF in the1020% range and low-normal levels of HbA2.

()0 Thalassemia

This condition has not been observed in the homozygousstate, presumably because it would be incompatible withfetal survival. Heterozygotes may be quite severely anemic atbirth, with the clinical picture of hemolytic disease of thenewborn25 associated with hypochromic red cells and globinchain imbalance typical of -thalassemia trait. For reasonsthat are not understood, anemia improves with age, hemo-globin level increasing during childhood; in adult life a bloodpicture typical of -thalassemia trait but with a normal HbA2level is observed.

The thalassemias

Homozygous 0 thalassemia

This condition, Hb Barts hydrops syndrome, is usually char-acterized by death in utero.1,12 These babies are either stillbornnear term or, if liveborn, untreated usually only survive for ashort period. The clinical picture is typical of hydrops fetalis,with marked edema and hepatosplenomegaly. The bloodpicture shows a hemoglobin level in the 68 g/dL range andthe red cells are hypochromic with numerous nucleatedforms. The hemoglobin consists of approximately 80% HbBarts, with the remainder the embryonic Hb Portland. Thissyndrome is associated with a high frequency of toxemia ofpregnancy, with postpartum bleeding, and other problemsdue to massive hypertrophy of the placenta. Autopsy studiesshow an increased frequency of fetal abnormalities, althoughthese are not always present; in a few babies that have beenrescued by exchange transfusion and maintained on regularred cell transfusions, growth and development has not alwaysbeen normal.

0/+ Thalassemia: HbH disease

This condition is characterized by a moderate degree of ane-mia and splenomegaly. It has a remarkably variable clinicalcourse: while some patients become transfusion dependent,

Fig. 13.7 Child with HbE thalassemia showing the typical facialappearance of the more severe forms of the disease. This child had

undergone splenectomy for worsening anemia.

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the majority are able to grow and develop normally withouttransfusion. The blood picture shows typical thalassemic redcell changes and the hemoglobin pattern is characterized byvariable amounts of HbH, small amounts of Hb Barts, and a low-normal level of HbA2. HbH may be demonstrated byincubating the red cells with a redox agent like brilliant cresylblue, which causes it to precipitate with the formation ofinclusion bodies. After splenectomy, large preformed inclu-sion bodies are present in many of the red cells.

-Thalassemia trait

The clinical picture of -thalassemia trait may result from the heterozygous state for 0 thalassemia ( /) or thehomozygous state for + thalassemia ( / ) as describedabove. These conditions are asymptomatic and the hemato-logic findings are characterized by a mild hypochromic anemia with a marked reduction in MCH and MCV. Thehemoglobin pattern is normal and these conditions can only be diagnosed with certainty by DNA analyses. In thenewborn period there are increased levels of Hb Barts, in the 510% range, but HbH is not demonstrable in adult life; occasional inclusions may be seen in the red cells in 0-thalassemia carriers.

Silent -thalassemia carriers

The heterozygous state for + thalassemia ( /) is associated with no hematologic abnormalities and a normalhemoglobin pattern in adult life. At birth approximately 50%of cases have slightly elevated levels of Hb Barts in the 13%range, but its absence does not rule out the diagnosis.

Thalassemia mental retardation syndromes

The ATR-16 syndrome is characterized by moderate mentalretardation and a very mild form of HbH disease or a bloodpicture resembling -thalassemia trait. Patients with this dis-order should undergo detailed cytogenetic analysis; in somecases chromosomal translocations may be found that are ofimportance for genetic counseling for future pregnancies. TheATR-X syndrome is characterized by severe mental retarda-tion, seizures, an unusual facial appearance with flattening ofthe nose, urogenital abnormalities, and other dysmorphicfeatures.26 The blood picture shows a mild form of HbH dis-ease or -thalassemia trait, and HbH inclusions can usuallybe demonstrated. The mothers of these children usually havesmall populations of red cells that contain HbH inclusions.

Screening and prevention

There are two major approaches to the avoidance of the thalassemias. Since the carrier states for thalassemia can

be easily recognized, it is possible to screen populations andprovide genetic counseling about the choice of marriage part-ners. If two -thalassemia heterozygotes marry, one in four oftheir children will have the severe compound heterozygousor homozygous disorder. Alternatively, when heterozygousmothers are identified prenatally, the husbands may betested; if they are also carriers, the couple may be counseledand offered the possibility of prenatal diagnosis and termina-tion of pregnancies carrying a fetus with a severe form of thalassemia.

Screening

If populations wish to offer marital choice, it is essential to develop premarital screening programs, best carried out in schoolchildren. It is vital to have a very well organizedgenetic counseling program in place and to provide both verbal advice and written information about the results ofscreening. The alternative approach is to screen every womanof an appropriate racial background in early pregnancy.

Probably the most cost-effective way of screening for thalassemia is through red cell indices.27 If MCV and MCHvalues are found to be in the range associated with the carrierstates for thalassemia, a HbA2 estimation should be carriedout.5 This will be elevated in the majority of cases of tha-lassemia. If the HbA2 level is normal, it is essential to refer thepatient to a center that can analyze the -globin genes. It isimportant to distinguish between the homozygous state for+ thalassemia ( / ) and the heterozygous state for 0 thalassemia ( /); in the former case the patient is not at risk for having a baby homozygous for 0 thalassemia with its attendant obstetric risks. In those rare cases in which the blood picture resembles heterozygous thalassemia but the HbA2 level is normal and the -globin genes are intact, thedifferential diagnosis lies between a nondeletional form of thalassemia and a normal-HbA2 form of thalassemia. Theseconditions have to be distinguished by globin chain synthesisanalysis and further DNA studies.28 It is important, of course,to carry out routine hemoglobin electrophoresis in all thesecases to exclude a coexisting structural hemoglobin variant.

Prenatal diagnosis

Prenatal diagnosis of different forms of thalassemia can becarried out in several ways.28 It can be made by studies ofglobin chain synthesis in fetal blood samples obtained byfetoscopy at 1820 weeks gestation, although this approachhas now been largely replaced by fetal DNA analysis. DNA is usually obtained by chorionic villous sampling (CVS)between weeks 9 and 12 of gestation. There is a small risk offetal loss and of the production of fetal abnormalities follow-ing this approach.27

The diagnostic techniques used for DNA analysis afterCVS have changed rapidly over recent years.28 The first

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diagnoses were carried out by Southern blotting of fetalDNA, using either restriction fragment length polymorphisms(RFLPs) combined with linkage analysis, or direct detectionof mutations. More recently, following the development ofthe polymerase chain reaction (PCR), the identification ofthalassemia mutations in fetal DNA has been greatly facili-tated. For example, it can be used for the rapid detection ofmutations that alter restriction enzyme cutting sites. The par-ticular fragment of the -globin gene is amplified, after whichthe DNA fragments are digested with an appropriate enzymeand separated by electrophoresis; these fragments can bedetected by either ethidium bromide or silver staining ofDNA bands on gels, and radioactive probes are not required.

Now that the mutations have been identified in manyforms of and thalassemia, it is possible to detect themdirectly as the first-line approach to fetal DNA analysis. The development of PCR, combined with the availability of oligonucleotide probes to detect individual mutations, has opened up a variety of new approaches for improving the speed and accuracy of carrier detection and prenatal diagnosis. For example, a diagnosis can be made using hybridization of specific 32P-end-labeled oligonucleotides toan amplified region of the -globin genes dotted onto nylonmembranes. Since the -globin gene sequence of interest canbe amplified more than a million-fold, thus increasing theefficiencies of probe annealing, hybridization times can belimited to 1 hour, and the entire procedure can be carried outin 2 hours.

There are many variations on the PCR approach to pre-natal diagnosis. For example, in a technique called ARMS(amplification refractory mutation system), which is based on the observation that, in many cases, oligonucleotides for the 3 mismatched residue will, under appropriate con-ditions, not function as primers in the PCR, it is possible to construct two specific primers.29 The normal primer isrefractory to PCR on a mutant template DNA, while themutant sequence is refractory to PCR on normal DNA. Othermodifications of PCR involve the use of nonradioactivelylabeled probes.

The error rate using these different approaches in most laboratories is now well below 1%.30 Potential sources of errorinclude maternal contamination of fetal DNA, nonpaternityand, if RFLP linkage analysis is used, genetic recombination.

More recently, other approaches have been developed forprenatal detection of the thalassemias, including the isolationof fetal cells from maternal blood and preimplantation diag-nosis.1 Although there have been a few reported successes of the prenatal diagnosis of thalassemia and Hb Lepore thalassemia by the analysis of fetal cells isolated from thematernal circulation, this technique is still under develop-ment and is only applicable to certain forms of thalassemia.Several laboratories have reported success with preimplanta-tion diagnosis but this remains technically difficult; this seemsto be a promising area for future development in this field.

Clinical management

Over recent years there have been major improvements in theclinical management of the severe forms of thalassemia.22

The development of better blood transfusion regimens, com-bined with effective iron-chelating therapy, has transformedthe outlook for children with this disease, at least those forwhom, in richer countries, this treatment is available.

Red cell transfusions

Regular red cell transfusions eliminate the complications of anemia and ineffective erythropoiesis, permit normalgrowth and development throughout childhood, and extendsurvival in thalassemia major.20,22 The decision to initiate regular transfusions is generally based on the observation of a hemoglobin concentration less than 6 g/dL over threeconsecutive months, a finding usually associated with poorgrowth, splenic enlargement and some degree of marrowexpansion. Determination of the molecular basis for severe thalassemia is only occasionally of value in predicting arequirement for regular transfusions.1,19 Prior to the firsttransfusion, iron and folate status should be assessed, a hepatitis B vaccine series should be initiated, and a completered cell phenotype obtained so that subsequent alloimmun-ization may be detected.

It is important that the pretransfusion hemoglobin con-centration does not generally exceed 9.5 g/dL.31 The pre-transfusion hemoglobin concentration, the volume of redcells administered, the weight of the patient, and the size ofthe spleen should be recorded at each visit in order to detect the development of hypersplenism.

Type of red cell concentrates

All patients should receive leukocyte-reduced red cell pre-parations.32 Early clinical experience with neocytes, youngred blood cells separated from older cells by density centri-fugation, suggested that their use is associated with modestextensions of transfusion intervals and reduction in annualtransfusional iron load.33 However, because these potentialbenefits are offset by major increases in preparation expensesand donor exposure,34 neocytes have had a minor impact onthe long-term management of patients with thalassemia.

Alloimmunization

Red cell alloimmunization may be higher in patients inwhom blood transfusion was begun after the age of 1 year,32

and lower in Mediterranean patients (310%) compared withAsian individuals (~21%), because of differences in antigenicdistribution.35 If possible, patients with thalassemia shouldreceive blood matched for ABO, CcDdEe, and Kell antigens.

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Splenectomy

In the past, an increase in transfusion requirements due tohypersplenism was frequently observed at the age of approx-imately 10 years. When annual transfusion requirementsexceed 200250 mL packed cells per kilogram body weight,splenectomy significantly reduces these requirements.36

In the modern era, with improved transfusion practices,hypersplenism is reduced and many patients do not requiresplenectomy.22 Concerns that splenectomy may be associ-ated with acceleration of iron loading in other organs remainunproven; the spleen does not appear to be a significantrepository of transfused iron.37 The biliary tract should becarefully assessed at surgery, but in the absence of diseasethere seems little place for prophylactic cholecystectomy.Because of the risk of postsplenectomy infection, splenec-tomy should usually be delayed until the age of 5 years.22 Atleast 3 weeks prior to splenectomy, patients should be vaccin-ated with the pneumococcal and Haemophilus influenzae typeB vaccines, and after surgery daily prophylactic penicillinshould be administered at least during childhood and prob-ably indefinitely. Erythromycin should be substituted forthose who are allergic to penicillin.

Infectious complications

Hepatitis B and hepatitis C virus, HIV, human T-cell leukemia virus and cytomegalovirus may cause significant morbidityand mortality in humans.32 Iron-induced hepatic damagemay be influenced by infection with hepatitis C virus, themost frequent cause of hepatitis in thalassemic children.The clinical and pathologic responses to interferon- in tha-lassemia patients with hepatitis C may be inversely related tobody iron burden.38 A 70% response rate in interferonnaivepatients treated for 12 months with interferon oca and rib-avirin has been reported.39 Infection with Yersinia enterocolit-ica, which poses a risk because of its growth enhancement iniron-rich environments,40 should be suspected in patientswith iron overload who present with high fever and no obvi-ous focus of infection. Even in the absence of a positive bloodculture for Yersinia in this clinical setting, therapy with intra-venous gentamicin and oral trimethoprim-sulfamethoxazoleshould be promptly instituted, and continued for at least 7days. Transfusion-transmitted malaria remains a major haz-ard in many developing countries.

Iron overload and iron-chelating therapy

Iron overload is the most important consequence of life-saving transfusions in thalassemia.20 The only chelatingagent approved for first-line therapy is deferoxamine. Thestandard method of deferoxamine administration today isprolonged parenteral infusion using portable ambulatorypumps (reviewed in ref. 20). Even suboptimal doses of defer-

oxamine prevent a rise in body iron during transfusion.41

Early reports of the beneficial effects of deferoxamine therapyon survival42,43 were confirmed by two studies from four NorthAmerican centers with follow-up periods of 1015 years.23,24

Patients in whom most serum ferritin concentrations weremaintained at less than 2500 g/L over 15 years had an estimated cardiac disease-free survival of 91%. Similarly,patients with hepatic storage iron less than 15 mg/g liver dry weight were protected from cardiac disease, impairedglucose tolerance and diabetes mellitus. Survival in recentstudies in compliant patients is reported as 95100% at 30 years of age.4449

Liver disease

The beneficial effects of deferoxamine on liver disease, thesecond leading cause of death in thalassemia,42 includereduction in iron concentration and improvement in liverfunction even in patients with massively elevated hepaticiron concentrations.41

Endocrine function

Long-term deferoxamine appears to have a beneficial effecton growth, sexual maturation and other endocrine complica-tions (reviewed in ref. 20). Normal pubertal development has been reported in approximately 5070% of youngerpatients,42 although the development of secondary amen-orrhea may develop eventually in about 20% of women.Adequate deferoxamine therapy also reduces the likeli-hood of the development of diabetes mellitus and hypo-thyroidism.24,44 A striking increase in fertility in both menand women, as well as many successful pregnancies, hasbeen reported over the last decade.45

Growth

In well-transfused but inadequately chelated patients, thefirst evidence of iron-mediated damage to the hypothalamicpituitary axis may be amelioration of the pubertal growthspurt, associated with delayed sexual maturation. In somepatients overintensive deferoxamine administration may bea cause of poor growth.46

Optimal body iron in patients with thalassemia major

The magnitude of the body iron burden is the principal deter-minant of clinical outcome.23,24 A balance must be struckbetween the risk of complications from iron overload and thetoxicity of deferoxamine, which is increased in the presenceof a relatively reduced body iron burden.47 A conservativegoal for iron-chelating therapy in thalassemia is maintenance

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of hepatic storage iron between 3.2 and 7 mg/g liver dryweight (Fig. 13.8).20,48

Management of iron-chelating therapy

One practical problem associated with long-term chelatingtherapy is the assessment of body iron. While plasma orserum ferritin concentration is the most commonly used indirect estimate of body iron stores, a concentration exceed-ing 4000 g/L, the result of ferritin release from damaged cells,may not reflect body iron stores.49 Changes above this level of serum ferritin may have limited clinical relevance. Inter-

pretation of the serum ferritin may be complicated by a vari-ety of conditions that alter concentrations independently ofchanges in body iron, including ascorbate deficiency, acuteand chronic infection and inflammation, hepatic disease, andineffective erythropoiesis.20 Because of this, in thalassemiamajor, variations in body iron account for only 57% of thevariation in serum ferritin.50

In contrast, measurement of hepatic iron concentration is a quantitative, specific and sensitive method of assessingbody iron burden in thalassemia (Table 13.2).51,52 Liver biopsyrepresents the best means of evaluating the pattern of ironaccumulation, and the inflammatory activity and histology of the liver. Liver biopsy under ultrasound guidance has alow complication rate in children,53 and should be performedat important crossroads in the management of the child withthalassemia.

Imaging techniques including computed tomography and magnetic resonance imaging (MRI) have been used toevaluate tissue iron stores in vitro and in vivo.51 Although MRIpotentially provides the best available technique for examin-ing the distribution of excess iron, further research is neededto develop means of making these measurements quantita-tive.51 To date, MRI has been more useful as a screening tech-nique for the detection of marked iron overload than as ameans for quantitative measurement.51 A calibration curverelating liver MRI signal to hepatic iron concentration has

Threshold for cardiac disease and early death

250

200

150

Hep

atic

iron

(m

ol/g

wet

wei

ght)

Hep

atic

iron

(mg/

g dr

y w

eigh

t)

100

50

0

50

40

30

20

10

0

Increased risk of complications

Optimal level for thalassemia major

Normal

Fig. 13.8 Thresholds of hepatic iron concentration in thalassemia (see text).

Table 13.2 Assessment of body iron burden in thalassemia.

Test Comments

Indirect (most tests widely available)

Serum/plasma ferritin concentration Noninvasive

Lacks sensitivity and specificity

Poorly correlated with hepatic iron concentration in individual patients

Serum transferrin saturation Lacks sensitivity

Tests of 24hour deferoxamine-induced Less than half of outpatient aliquots collected correctly

urinary iron excretion Ratio of stool to urine iron variable; poorly correlated with hepatic iron concentration

Imaging of tissue iron

Computed tomography: liver Variable correlation with hepatic iron concentration reported

Magnetic resonance

Liver Variable correlations with hepatic iron concentration reported

Treatment-induced changes confirmed by liver biopsy

Heart Only modality available to image cardiac iron stores; changes observed during chelating therapy

consistent with reduction in cardiac iron

Anterior pituitary Only modality available to image pituitary iron; signal moderately well correlated with pituitary reserve

Evaluation of organ function Most tests lack sensitivity and specificity; may identify established organ dysfunction

Direct (most tests not widely available)

Cardiac iron quantitation: biopsy Imprecise due to inhomogeneous distribution of cardiac iron

Hepatic iron quantitation: biopsy Reference method; provides direct assessment of body iron burden, severity of fibrosis and inflammation

Safe when performed under ultrasound guidance

Superconducting susceptometry (SQUID) Noninvasive; excellent correlation with biopsy-determined hepatic iron

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been deduced over a clinically relevant range of hepatic iron concentrations, with a coefficient of variation of 2.1%.54

Biomagnetic susceptometry (SQUID)55 provides the onlynoninvasive measurement of tissue iron stores validated foruse in clinical studies, but it is available in only a few centersworldwide.51 Recently, attempts to assess cardiac iron usingdifferent MRI techniques have included a much-promotedT2* technique,56,57 the validity and prognostic significanceof which requires clarification by long-term, prospectivestudies.58 The most comprehensive and valid assessment of cardiac function is still the combined information obtained from the patients transfusion record, knowledge of adherence to a chelation regimen, and serial hepatic ironconcentrations.59

Initiation and management of chelating therapy

Few guidelines exist with respect to the start of iron-chelatingtherapy. In practice, the usual approach is to begin a regimenof nightly subcutaneous deferoxamine based on the serumferritin concentration after a period of regular transfusions,but initiation (and titration) of deferoxamine therapy is ideally based on hepatic iron concentration, determined after 1 year of regular transfusions. If a liver biopsy is not obtained,treatment with subcutaneous deferoxamine not exceeding2535 mg/kg body weight per 24 hours should be initiatedafter approximately 1 year of regular transfusions.

Balance between effectiveness and toxicity ofdeferoxamine (Table 13.3)

Virtually all toxicities are dose related, occurring only whenexcessive doses, or regular doses in patients with modest bodyiron burdens, are administered. Guidelines for safe dosageinclude restricting daily doses to 50 mg/kg body weight orless and twice-yearly calculation of a toxicity index, calcu-lated as mean daily dose of deferoxamine (mg/kg) dividedby serum ferritin concentration (g/L), which should notexceed 0.025.47,60,61

Alternatives to subcutaneous infusion of deferoxamine

The most common difficulty associated with long-term therapyis erratic compliance, especially in adolescents. Regimens of intravenous ambulatory deferoxamine, in which drug isadministered through implantable venous access ports,62 cir-cumvents tissue irritation and are associated with rapid reduc-tion of body iron burden and good patient compliance. Suchregimens have been shown to reverse cardiac dysfunction.63

Orally active iron-chelating agents

The expense and inconvenience of deferoxamine has led to along-time search for an orally active iron chelator. The agentmost extensively evaluated to date is deferiprone. Clinical

Table 13.3 Monitoring of deferoxamine-related toxicity.

Toxicity

High-frequency

sensorineural

hearing loss

Retinal abnormalities

Metaphyseal and spinal

abnormalities

Decline in height velocity

and/or sitting height

DFO, deferoxamine; HIC, hepatic iron concentration.

Investigations

Audiogram

Retinal examination

Radiography of wrists,

knees, thoraco-lumbar-

sacral spine

Bone age of wrist

Determination of sitting

and standing heights

Frequency

Yearly; if patient symptomatic,

immediate reassessment

Yearly; if patient symptomatic,

immediate reassessment

Yearly

Twice yearly

Alteration in therapy

Interrupt DFO immediately

Directly assess body iron burden

Discontinue DFO 6 months if HIC 3.27 mg/g dry wt liver tissue

Repeat audiogram every 3 months until normal or stable

Adjust DFO to HIC (see Table 15.2)

Interrupt DFO immediately

Directly assess body iron burden

Discontinue DFO 6 months if HIC 3.27 mg/g dry wt liver tissueReview every 3 months until normal or stable

Adjust DFO to HIC (see Table 15.2)

Reduce DFO to 25 mg/kg daily 4/weekDirectly assess body iron burden

Discontinue DFO 6 months if HIC 3 mg/g dry weight liver tissueReassess HIC after 6 months

Adjust DFO to HIC (see Table 15.2)

As for metaphyseal and spinal abnormalities

Regular (6-monthly) assessment by pediatric endocrinologist

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trials that were intended to support an application for thelicensing of deferiprone were terminated prematurely bytheir corporate sponsor in 1996 after concerns were raised by the investigators about inadequate control of body iron inmany patients.64 Because serum ferritin showed a less clearrelationship to hepatic iron concentration65 than in patientstreated with deferoxamine, it may be problematic to interpretchanges in serum ferritin as evidence of the effectiveness ofdeferiprone. In studies which determined hepatic storageiron, this exceeded 15 mg/g dry weight in 1865% of patientstreated over 28 years,66 raising concerns that deferipronemay not be sufficiently effective to protect all patients fromiron-induced cardiac disease.

Claims that deferiprone is a more effective cardiopro-tective agent than deferoxamine are not yet supported byprospective clinical data.58,67 Continued cardiac mortality inpatients receiving deferiprone has been reported,65,68 includ-ing deaths after years of treatment, during which period animprovement in cardiac disease is regularly observed withdeferoxamine.

Since 1999, deferiprone has been licensed for patients in Europe unable to use deferoxamine. An application submitted to North American regulators in 2002 has beenunsuccessful in obtaining marketing approval to date.Reassuringly, only 16 patients truly unable to use defer-oxamine are reported in the literature over 20 years;69 all have been capable of being desensitized and of resumingdeferoxamine safely. Non-compliance remains a seriousproblem however.

Deferiprone has also been administered in combinationwith deferoxamine (although this combination is not formallypermitted under European licensing). Most of these areshort-term studies, with periods of administration less than 2years, report poor control of iron in most of the 39 patients.No long-term efficacy or safety data are available for com-bination therapy.69a

Toxicity of deferiproneReports of arthropathy in deferiprone-treated patients aresomewhat confusing. Most reviews claim that this complica-tion, varies between ethnic groups; subtotal destructionwith an incidence of 210%, is reversible on stopping thedrug. However, recent data suggest that both the incidenceand severity of this complication varies between ethnicgroups; subtotal destruction of the knee joints has now beenobserved in some patients. In India,70,71 Sri Lanka,72,73 andLebanon,74,75 arthropathy is reported as developing in up to50% of patients. A lower incidence (018%) has been reportedfrom other centers.68,7680

Accelerated progression of hepatic fibrosis has beenobserved at proportions of 29%,78 36%,81 37%,79 and 0%82 (ofall patients who underwent baseline and follow-up biopsies).In some79,82 but not all81 studies, liver function worsened in

deferiprone-treated patients, regardless of hepatitis C virusstatus, while remaining unchanged in deferoxamine-treatedpatients. More worrisome is the evidence of hepatocellulardamage in deferiprone-treated patients emerging after manyyears of treatment,68,83 consistent with the observation thatthe time to onset of accelerated hepatic damage might exceed 3 years.81 The one study to report no accelerated liver dam-age82 was undermined by a failure to report striking increases(3285% over baseline) in hepatocellular iron, although this finding was reported in an earlier publication.84 Othertoxicities of deferiprone include nausea, agranulocytosis and neutropenia. In summary, it is generally accepted thatdeferiprone is less effective than deferoxamine in maintain-ing iron balance. Its potential for cardioprotection requiresfurther prospective study.85

Other orally active iron chelatorsThe iron chelator ICL670A (Exjade; Novartis) has undergoneearly evaluation in a randomized, double-blind, placebo-controlled, dose-escalation study that established its short-term tolerability.86 This was followed by Phase II and PhaseIII studies to examine short-term efficacy and safety,8789 inwhich hepatic iron was assessed at baseline and after 1 yearof treatment. The long-term effectiveness and safety of thisdrug await further study.

Bone marrow transplantation

Allogeneic bone marrow transplantation offers an acceptedalternative to standard clinical management. Three pretrans-plant characteristics are significantly associated with survivaland event-free survival: (i) hepatomegaly greater than 2 cmbelow the costal margin; (ii) the presence of portal fibrosis on liver biopsy; and (iii) the effectiveness of therapy withdeferoxamine prior to transplantation.90 In patients in whomnone of these factors were present prior to transplantation(identified as class 1 patients), event-free survival exceeded90%; in contrast, in those with all three (class 3 patients),event-free survival was only 56%. In over 1000 consecutivetransplants in patients aged 135 years, overall survival was68%.91 In class 1 patients, thalassemia-free survival was 90%and survival was 93%. Umbilical cord blood has been used asa source of stem cells for class 1 and 2 children.92

Less than 30% of patients have an available HLA-identicalfamily donor. In a study of unrelated donors matched by high-resolution molecular HLA methods (extended haplotypes),69% of patients were alive and well after 7109 months.93

This approach, and the use of related partially mismatcheddonors, is considered experimental in thalassemia.94

Successful transplantation liberates patients from chronictransfusions, but does not eliminate the necessity for iron-chelating therapy in all cases. Iron overload and hepatitis

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C virus are independent and synergistic risk factors for pro-gression of hepatic fibrosis and the development of cirrhosisin the ex-thalassemic. Removal of iron by phlebotomy anddeferoxamine is safe and effective in the reduction of tissueiron and the arrest of fibrosis and early cirrhosis.95

Many parents may be confronted with the choice betweenstandard medical (transfusion and chelation) therapy andbone marrow transplantation. The excellent results from Italy suggest that marrow transplantation should be offeredto any patient with a compatible donor. On the other hand,the choice between these therapeutic approaches may be adifficult one, since extended cardiac disease-free survival in patients regularly compliant with deferoxamine exceeds90%, comparable to that achieved with transplantation inclass 1 patients. However, while the long-term outcomes oftransplantation are not fully known, and mortality is alwaysa risk, this cost-saving procedure renders patients not merelycardiac disease-free but also thalassemia-free. Furthermore,if compliance with regular deferoxamine falters or ineffectivetreatments are embarked upon, survival in medically treatedpatients may be considerably less than 90%.

Experimental approaches

Augmentation of fetal hemoglobin synthesis may reduceglobin chain imbalance and ameliorate the severity of the dis-ease.96 Several cell cycle-specific chemotherapeutic agents,and nonchemotherapeutic drugs including hematopoieticgrowth factors and short-chain fatty acids, stimulate HbFproduction in vitro and in animal models. Clinical trials inthalassemia have included short- and long-term administra-tion of 5-azacytidine, hydroxycarbamide, recombinant humanerythropoietin, butyric acid compounds, and combinationsof these agents.96

5-Azacytidine successfully increased HbF levels in earlystudies of patients with thalassemia but predictably causesbone marrow suppression, limiting drug administration insome patients. Consideration of its potential adverse effectsshifted interest to the use of alternate therapies.96,97

Clinical responses associated with administration of hy-droxycarbamide have been disappointing, given the recentobservation that patients with certain polymorphisms andspecific mutations, notably Gy-158 (c T) and Hb Lepore,may demonstrate durable responses to hydroxycarbamide,with some patients achieving transfusion-independence.97

Further trials may be of interest.In patients treated with recombinant human erythropoie-

tin therapy, total hemoglobin increased in a few, usually with-out observed effects on HbF synthesis.96,97 This expensiveregimen has not shown great promise in thalassemia.

Butyric acid compounds, derivatives of natural short-chain fatty acids, have offered potential therapy for the

hemoglobinopathies following the observation that elevatedplasma concentrations of -amino-n-butyric acid in infants of diabetic mothers delayed the switch from to globinaround the time of birth.98 Hematologic responses to argininebutyrate and sodium phenylbutyrate have been reported inthalassemia,97 the most striking, for reasons that remainundefined,99 in two siblings with homozygous Hb Lepore.

Management of other forms of thalassemia

Thalassemia intermedia

Thalassemia intermedia, a descriptive title with no clear-cutgenetic meaning, is used to refer to patients with a hemo-globin level persistently below 9 g/dL who can be main-tained without transfusions. The diagnosis should be madeonly after a considerable period of observation, and oftenrequires revision.

At diagnosis, folate supplementation should be initiatedand the hemoglobin should be determined twice monthly. A substantial decline should prompt investigations for secondary causes, including infection, folate deficiency, orhypersplenism. In patients over 4 years of age, splenectomymay be indicated prior to initiation of a program of regulartransfusions. Some patients may become less tolerant of ane-mia with advancing age, or may develop transfusion depend-ency in adolescence or early adulthood. Abnormal growth,pathologic fractures, or signs of intolerance of anemia shouldprompt consideration of a program of regular transfusions.Spinal or nerve compression should be treated by red celltransfusion or local irradiation followed by transfusions.100

Iron loading is less accelerated than in thalassemia major. Daily gastrointestinal iron loading may be in the orderof 39 mg, or about 25 g/year.101 In contrast, in regularlytransfused patients iron accumulation is in the range of 67 g/year. The coinheritance of hereditary hemochromatosismay adversely increase iron loading in thalassemia inter-media.102 In patients with elevated serum ferritin concentra-tions, assessment of liver iron is indicated; if this exceeds 6 mg/g liver dry weight, deferoxamine should be initiated.

HbE thalassemia

This is the commonest form of severe thalassemia in manyAsian countries and is being seen increasingly in NorthAmerica and Europe. Relatively little is known about its natural history, the reasons for its clinical diversity, or how it should be managed and it may not be possible to define its severity without a long period of observation. Until moreis known about this important disease, approaches to itsmanagement should follow those recommended above forthalassemia intermedia.

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The thalassemias

All children with Hb Barts hydrops fetalis syndrome requireregular blood transfusion and chelation therapy. Many patientswith HbH disease can be managed conservatively with regu-lar folate supplementation and avoidance of oxidant drugs,which tend to exacerbate their anemia. Occasionally, thehemoglobin level is such that a regular transfusion regimen,similar to that employed for severe cases of thalassemia,may be required. Overall, the results of splenectomy are dis-appointing, and in a few cases it has been followed by severethrombotic complications. Older patients may accumulateconsiderable amounts of iron through increased gastro-intestinal absorption. Coexistence with the common form ofhereditary hemochromatosis may explain more severe ironoverload in occasional patients.

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