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CHAPTER 46 THE THALASSEMIAS

CHAPTER 46 THE THALASSEMIAS
Williams Hematology

CHAPTER 46 THE THALASSEMIAS

DAVID J. WEATHERALL

Definitions and History

Different Forms of Thalassemia
Etiology and Pathogenesis

Genetic Control and Synthesis of Hemoglobin

Globin Gene Clusters

Molecular Basis of the Thalassemias
Pathophysiology

Imbalanced Globin-Chain Synthesis

Persistent Fetal Hemoglobin Production and Cellular Heterogeneity

Consequences of Compensatory Mechanisms for the Anemia of Thalassemia

Splenomegaly: Dilutional Anemia

Abnormal Iron Metabolism

Coagulation Defects

Clinical Heterogeneity
Population Genetics
Clinical Features

b and db Thalassemias

a Thalassemias
Laboratory Features

b Thalassemia Major

b Thalassemia Minor

a Thalassemias
Differential Diagnosis

Common Forms of Thalassemia

Less Common Forms of Thalassemia
Therapy, Course, and Prognosis

Transfusion

Iron Chelation

Marrow Transplantation

Therapies of Special Types of Thalassemia

Experimental Approaches to Treatment

Prognosis
Prevention
Chapter References

The thalassemias are the commonest monogenic diseases in Man. They occur at a high gene frequency throughout the Mediterranean populations, the Middle East, the Indian subcontinent, and Burma and in a line stretching from southern China through Thailand and the Malay peninsula into the island populations of the Pacific. They are also seen commonly in countries in which there has been immigration from these high-frequency populations.
There are two main classes of thalassemia, a and b, in which the a- and b-globin genes are involved, and rarer forms due to abnormalites of other globin genes. These conditions all have in common an imbalanced rate of production of the globin chains of adult hemoglobin, a chains in b thalassemia and b chains in a thalassemia. Several hundred different mutations at the a- and b-globin loci have been defined as the cause of the reduced or absent output of a or b chains.
The pathophysiology of the thalassemias can be traced to the deleterious effects of the globin-chain subunits that are produced in excess. In b thalassemia, excess a chains cause damage to the red cell precursors and red cells and lead to profound anemia. This causes expansion of the ineffective marrow, with severe effects on development, bone formation, and growth. The major cause of morbidity and mortality is the effect of iron deposition in the endocrine organs, liver, and heart, which results from increased intestinal absorption and the effects of blood transfusion. The pathophysiology of the a thalassemias is different because the excess b chains that result from defective a-chain production form b4 molecules, or hemoglobin H, which is soluble and does not precipitate in the marrow. However, it is unstable and precipitates in older red cells. Hence, the anemia of a thalassemia is hemolytic rather than dyserythropoietic.
The clinical pictures of a and b thalassemia vary widely, and knowledge is gradually being amassed about some of the genetic factors that modify these phenotypes.
Since the carrier states for the thalassemias can be identified and affected fetuses can be diagnosed by DNA analysis after the ninth to tenth week of gestation, these conditions are widely amenable to prenatal diagnosis. Their symptomatic management is based on regular blood transfusion, iron chelation therapy, and the judicious use of splenectomy. Current experimental approaches to their management include the stimulation of fetal hemoglobin synthesis and attempts at somatic cell gene therapy.

Acronyms and abbreviations that appear in this chapter include: bp, base pairs; HPFH, hereditary persistence of fetal hemoglobin; LCR, locus control region; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

DEFINITIONS AND HISTORY
A form of severe anemia occurring early in life and associated with splenomegaly and bone changes was first described by Cooley and Lee in 1925.1 In 1932, George H. Whipple and William L. Bradford published a comprehensive account of the pathologic findings in this disease.2 Whipple coined the phrase thalassic anemia,3,4 condensing it to thalassemia, from qaiassa, “the sea,” since early patients were all of Mediterranean background. It was only after 1940 that the true genetic character of this disorder was fully appreciated. It became clear that the disease described by Cooley and Lee is the homozygous state of an autosomal gene for which the heterozygous state is associated with much milder hematologic changes. The severe homozygous condition became known as thalassemia major, while the heterozygous states, thalassemia trait, were designated, according to their severity, thalassemia minor or minima.3,5,6,7 and 8 Later, the term thalassemia intermedia was used to describe disorders that are milder than the major form but more severe than the traits.
More recently it has been established that thalassemia is not a single disease but a group of disorders, each of which results from an inherited abnormality of globin production.7 These conditions form part of the spectrum of diseases known collectively as the hemoglobinopathies, which can be classified broadly into two types. First, there are those, such as sickle cell anemia, that result from an inherited structural alteration in one of the globin chains. Although such abnormal hemoglobins may be synthesized less efficiently or broken down more rapidly than normal adult hemoglobin, the associated clinical abnormalities result from the physical properties of the abnormal hemoglobin (see Chap. 48). The second major subdivision of the hemoglobinopathies, the thalassemias, is constituted by inherited defects in the rate of synthesis of one or more of the globin chains. This causes imbalanced globin chain production, ineffective erythropoiesis, hemolysis, and a variable degree of anemia.
Several monographs describe the historical aspects of thalassemia in more detail.3,5,7
DIFFERENT FORMS OF THALASSEMIA
Thalassemia can be defined as a condition in which a reduced rate of synthesis of one or more of the globin chains leads to imbalanced globin-chain synthesis, defective hemoglobin production, and damage to the red cells or their precursors from the effects of the globin subunits that are produced in relative excess.3,7,8 and 9 The main varieties of thalassemia that have now been defined with certainty are summarized in Table 46-1.

TABLE 46-1 THE THALASSEMIAS AND RELATED DISORDERS

The b thalassemias can be divided into two main varieties: in one form, b0 thalassemia, there is a total absence of b-chain production, and in the other, b+ thalassemia, there is a partial deficiency of b-chain production. The hallmark of the common forms of b thalassemia is an elevated level of hemoglobin A2 in heterozygotes. However, there is a less common class of b thalassemias in which heterozygotes have normal levels of hemoglobin A2. These conditions are sometimes subdivided into type 1, in which there are no hematological abnormalities and hence the condition is “silent” in the heterozygous state, and type 2, in which the hematological changes are indistinguishable from those of b-thalassemia heterozygotes with elevated hemoglobin A2 levels.
The db thalassemias are also heterogeneous. In some cases, no d or b chains are synthesized. Originally it was customary to classify these disorders according to the structure of the hemoglobin F produced, that is, GgAg(db)0 and Gg(db)0 thalassemia. In fact, this is illogical, and these conditions are best described by the globin chains that are defectively synthesized, that is, simply into the (db)+, (db)0, and (Agdb)0 thalassemias.7,10 In the (db)+ thalassemias an abnormal hemoglobin is produced that has normal a chains combined with non-a chains that consist of the N-terminal residues of the d chain fused to the C-terminal residues of the b chain. These fusion variants, called the Lepore hemoglobins, also show structural heterogeneity.
The d thalassemias7,10 are characterized by a reduced output of d chains and hence by reduced levels of hemoglobin A2 in heterozygotes and an absence of hemoglobin A2 in homozygotes. They are of no clinical significance.
A disorder characterized by defective e, g, d, and b-chain synthesis has been defined at the clinical and molecular level.7,10 The homozygous state for this condition, egdb thalassemia, is presumably not compatible with fetal survival, and it has been observed only in heterozygotes.
To complete this description of the thalassemia-like mutations that involve the b-globin gene complex, we must consider hereditary persistence of fetal hemoglobin (HPFH).7,9,10 This heterogeneous condition is characterized by persistence of fetal hemoglobin production into adult life in the absence of major hematologic changes. It is classified into deletion and nondeletion forms. The deletion forms of HPFH can be classified, like db thalassemia, as (db)0 HPFH and then further subdivided according to the particular population in which this occurs and its associated molecular defect. In effect, the deletion forms of HPFH are very similar to db thalassemia except that there is more efficient g-chain synthesis and therefore less chain imbalance and a milder phenotype. However, the homozygous state is associated with mild thalassemic changes, and, in fact, the db thalassemias and deletion forms of HPFH form a clinical continuum. The nondeletion forms of HPFH are also heterogeneous. In some cases they are associated with mutations that involve the b-globin gene cluster and in which there is b-chain synthesis cis to the HPFH determinant. These conditions are subdivided into Ggb+ HPFH and Agb+ HPFH. Again, they are often subclassified according to the population in which they occur, for example, Greek HPFH, British HPFH, and so on. Finally, there is a heterogeneous group of HPFH determinants associated with very low levels of persistent fetal hemoglobin, the genetic loci of which, at least in some cases, are not linked to the b-globin gene cluster.
Since a chains are present in both fetal and adult hemoglobins, a deficiency of a-chain production will affect hemoglobin synthesis in fetal as well as adult life. A reduced rate of a-chain synthesis in fetal life results in an excess of g chains, which form g4 tetramers, or hemoglobin Bart’s. In adult life, a deficiency of a chains results in an excess of b chains, which form b4 tetramers, or hemoglobin H. Because there are two a-globin genes per haploid genome, the genetics of a thalassemia is more complicated than that of b thalassemia. There are two main groups of a-thalassemia determinants.7,10 First, there are the a0 thalassemias (formerly called a thalassemia 1), in which no a chains are produced from an affected chromosome. That is, both linked a-globin genes are inactivated. Second, there are the a+ thalassemias (formerly called a thalassemia 2), in which the output of one of the linked pair of a-globin genes is defective. The a+ thalassemias are subdivided into deletion and nondeletion types. Both the a0 thalassemias and deletion and nondeletion forms of a+ thalassemia are all extremely heterogeneous at the molecular level. There are two major clinical phenotypes of a thalassemia, the hemoglobin Bart’s hydrops syndrome, which usually reflects the homozygous state for a0 thalassemia, and hemoglobin H disease, which usually results from the compound heterozygous state for a0 and a+ thalassemia.
Since the structural hemoglobin variants and the thalassemias occur with a high frequency in some populations, the two types of genetic defect may be found in the same individual. The different genetic varieties of thalassemia and their combinations with the genes for abnormal hemoglobins produce a series of disorders known collectively as the thalassemia syndromes.7
ETIOLOGY AND PATHOGENESIS
GENETIC CONTROL AND SYNTHESIS OF HEMOGLOBIN9,10
The structure and ontogeny of the hemoglobins are reviewed in Chap. 29 and Chap. 8, respectively, and only those aspects with particular relevance to the thalassemia problem are restated here.
Human adult hemoglobin is a heterogeneous mixture of proteins consisting of a major component, hemoglobin A, and a minor component, hemoglobin A2, constituting about 2.5 percent of the total. In intrauterine life, the main hemoglobin is hemoglobin F. The structure of these hemoglobins is similar. Each consists of two separate pairs of identical globin chains. Except for some of the embryonic hemoglobins (see below), all the normal human hemoglobins have one pair of a chains: in hemoglobin A these are combined with b chains (a2b2), in hemoglobin A2 with d chains (a2d2), and in hemoglobin F with g chains (a2g2).
Human hemoglobin shows further heterogeneity, particularly in fetal life, and this has important implications for understanding the thalassemias and for approaches to their prenatal diagnosis. Hemoglobin F is a mixture of molecular species with the formulas a2g2136Gly and a2g2136Ala. The g chains containing glycine at position 136 are designated Gg chains; those that contain alanine are called Ag chains. At birth the ratio of molecules containing Gg chains to those containing Ag chains is about 3:1; this ratio varies widely in the trace amounts of hemoglobin F present in normal adults.
Before the eighth week of intrauterine life there are three embryonic hemoglobins: hemoglobins Gower 1 (z2e2), Gower 2 (a2e2), and Portland (z2g2). The z and e chains are the embryonic counterparts of the adult a and b and g and d chains, respectively. z-Chain synthesis persists beyond the embryonic stage of development in some of the a thalassemias; so far, persistent e-chain production has not been found in any of the thalassemia syndromes. During fetal development there is an orderly switch from z to a and from e- to g-chain production, followed by b- and d-chain production after birth.
The different human hemoglobins, together with the arrangement of the a-gene cluster on chromosome 16 and the b-gene cluster on chromosome 11, are shown in Fig. 46-1.

FIGURE 46-1 The genetic control of human hemoglobin. The main globin gene clusters are on chromosomes 11 and 16. At each stage of development different genes in these clusters are activated or repressed. The different globin chains directed by individual genes are synthesized independently and combine with each other in a random fashion as indicated by the arrows.

GLOBIN GENE CLUSTERS
Although there is some individual variability, the a-gene cluster usually contains one functional z gene and two a genes, designated a2 and a1. It also contains four pseudogenes: yx1, ya1, ya2, and q1.9,10 The latter is remarkably conserved among different species. Although it appears to be expressed in early fetal life, its function is unknown; it seems unlikely that it can produce a viable globin chain. Each a gene is located in a region of homology approximately 4 kb long, interrupted by two small nonhomologous regions.11,13 It is thought that the homologous regions have resulted from gene duplication and that the nonhomologous segments may have arisen subsequently by insertion of DNA into the noncoding regions around one of the two genes. The exons of the two a-globin genes have identical sequences. The first intron in each gene is identical, but the second intron of a1 is nine bases longer and differs by three bases from that in the a2 gene.13,14 and 15 Despite their high degree of homology, the sequences of the two a-globin genes diverge in their 3′ untranslated regions 13 bases beyond the TAA stop codon. These differences provide an opportunity to assess the relative output of the genes, an important part of the analysis of the a thalassemias.16,17 It appears that the production of a2 messenger RNA exceeds that of a1 by a factor of 1.5 to 3. x1 and x2 genes are also highly homologous. The introns are much larger than those of a-globin genes, and, in contrast to the latter, IVS-1 is larger than IVS-2. In each x gene, IVS-1 contains several copies of a simple repeated 14-bp sequence that is similar to sequences located between the two x genes and near the human insulin gene. There are three base changes in the coding sequence of the first exon of x1, one of which gives rise to a premature stop codon, thus making it an inactive pseudogene.
The regions separating and surrounding the a-like structural genes have been analyzed in detail. Of particular relevance to thalassemia is the fact that this gene cluster is highly polymorphic.18 There are five hypervariable regions in the cluster: one downstream from the a1 gene, one between the x and yx genes, one in the first intron of both the x genes, and one 5′ to the cluster. These regions have been found to consist of varying numbers of tandem repeats of nucleotide sequences. Taken together with the single-base restriction fragment length polymorphisms (RFLPs), the variability of the a-globin gene cluster reaches a heterozygosity level of approximately 0.95. Thus, it is possible to identify each parental a-globin gene cluster in the majority of persons. This heterogeneity has important implications for tracing the history of the thalassemia mutations.
The arrangement of the b-globin gene cluster on the short arm of chromosome 11 is shown in Fig. 46-1. Each of the individual genes and their flanking regions have been sequenced.19,20,21 and 22 Like the a1 and a2 gene pairs, the Gg and Ag genes share a similar sequence. In fact, the Gg and Ag genes on one chromosome are identical in the region 5′ to the center of the large intron yet show some divergence 3′ to that position. At the boundary between the conserved and divergent regions, there is a block of simple sequence that may be a “hot spot” for the initiation of recombination events that have led to unidirectional gene conversion.
Like the a-globin genes, the b-gene cluster contains a series of single-point RFLPs, although in this case no hypvariable regions have been identified.23,24 The arrangement of RFLPs, or haplotypes, in the b-globin gene cluster falls into two domains. On the 5′ side of the b gene, spanning about 32 kb from the e gene to the 3′ end of the yb gene, there are three common patterns of RFLPs. In the region encompassing about 18 kb to the 3′ side of the b-globin gene, there are also three common patterns in different populations. Between these regions there is a sequence of about 11 kb in which there is randomization of the 5′ and 3′ domains and hence where a relatively higher frequency of recombination may occur. The b-globin gene haplotypes are similar in most populations but differ markedly in individuals of African origin; these findings suggest that these haplotype arrangements were laid down very early during evolution, and they are consistent with data obtained from mitochondrial DNA polymorphisms that point to the early emergence of a relatively small population from Africa with subsequent divergence into other racial groups.25 Again, they are extremely useful for analyzing the population genetics and history of the thalassemia mutations.
The regions flanking the coding regions of the globin genes contain a number of conserved sequences that are essential for their expression.26 The first is the TATA box, which serves accurately to locate the site of transcription initiation at the CAP site, usually about 30 bases downstream, and also appears to influence the rate of transcription. In addition, there are two so-called upstream promotor elements; 70 or 80 base pairs (bp) upstream is a second conserved sequence, the CCAAT box, and further 5′, approximately 80 to 100 bp from the CAP site, is a CACCC homology box that can be either inverted or duplicated.7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 and 26 These promotor sequences are also required for optimal transcription, and, as we shall see later, mutations in this region of the b-globin gene cause its defective expression. The globin genes also have conserved sequences in their 3′ flanking regions, notably AATAAA, which is the polyadenylation signal site.
REGULATION OF GLOBIN GENE CLUSTERS
The mechanism of globin gene expression is summarized in detail in Fig. 46-2. In short, the primary transcript is a large mRNA precursor containing both intron and exon sequences. During its stay in the nucleus, it undergoes a good deal of processing that entails capping the 5′ end and polyadenylation of the 3′ end, both of which probably serve to stabilize the transcript (see Chap. 11). The intervening sequences are removed from the mRNA precursor in a complex two-stage process that relies on certain critical sequences at the intron-exon junctions.

FIGURE 46-2 The expression of a human globin gene.

The way in which the globin gene clusters are regulated is of major relevance to an understanding of the pathogenesis of the thalassemias. While many details remain to be worked out, studies carried out over the last few years have provided at least an outline of some of the major mechanisms of globin gene regulation.10,26,27
Most of the DNA within cells that is not involved in gene transcription is packaged into a compact form that is inaccessible to transcription factors and RNA polymerase. Transcriptional activity is characterized by a major change in the structure of the chromatin surrounding a particular gene. These alterations in chromatin structure can be identified by enhanced sensitivity to exogenous nucleases. Erythroid lineage–specific nuclease-hypersensitive sites are found at several locations in the b-globin gene cluster, which vary during different stages of development. In fetal life these sites are associated with the promoter regions of all four globin genes, while in adult erythroid cells the sites associated with the g genes are absent. The methylation state of the genes also plays an important role in their ability to be expressed; in human and other animal tissues, the globin genes are extensively methylated in nonerythroid organs and are relatively undermethylated in hematopoietic tissues. The changes in chromatin configuration around the globin genes at different stages of development are reflected by alterations in their methylation state.28
In addition to the promoter elements mentioned earlier, several other important regulatory sequences have been identified in the globin gene clusters. For example, several enhancer sequences have been identified that are thought to be involved with tissue-specific expression. Their sequences are similar to the upstream activating sequences of the promoter elements. Both consist of a number of “modules,” or motifs, that contain binding sites for transcriptional activators or repressors.29 The enhancer sequences are thought to act by coming into spatial apposition with the promoter sequences to increase the efficiency of transcription of particular genes. It is now clear that transcriptional regulatory proteins may bind both to the promoter region of a gene and to the enhancer. It appears that some of these transcriptional proteins, GATA-1 and NFE-2, for example, are restricted to hematopoietic tissues.30,31 These proteins may act to bring the promoter and the enhancer into close physical proximity, permitting transcription factors bound to the enhancer to interact with the transcriptional complex that forms near the TATA box. It seems likely that at least some of these hematopoietic gene transcription factors will turn out to be developmental stage specific.
Upstream from the embryonic globin genes in both the a- and b-gene clusters there is another set of erythroid-specific nuclease-hypersensitive sites. These mark the regions of particularly important control elements. In the case of the b-globin gene cluster, this region is marked by five hypersensitive sites.32 The most 5′ site (HS5) does not show tissue specificity, while HSs 1 through 4, which together form the locus control region (LCR), are largely erythroid specific. Each of the regions of the LCR contains a variety of binding sites for erythroid transcription factors. Although the precise function of the LCR is not known, it is undoubtedly required to establish a transcriptionally active domain spanning the entire globin gene cluster. The a-globin gene cluster also has a major regulatory element of this kind, in this case called HS40.33 Its structure closely resembles HS2 of the b-globin gene cluster LCR; a 350-bp core fragment retains most of the activity and contains a duplicated NF-E2 binding site flanked by GATA-1 sites. Although deletions of this region inactivate the entire a globin gene cluster, its action must be fundamentally different from that of the b-globin LCR, since the chromatin structure of the a gene cluster is in an open conformation in all tissues.
Some forms of thalassemia result from deletions that involve these regulatory regions. In addition, the phenotypic effects of deletions of these gene clusters are strongly positional, which may reflect the relative distance of particular genes from the LCR and HS40.
DEVELOPMENTAL CHANGES IN GLOBIN GENE EXPRESSION
One aspect of the human globin genes that is of particular importance is the regulation of the switch from fetal to adult hemoglobin. Since many of the thalassemias and related disorders of the b-globin gene cluster are associated with persistent g-chain synthesis, a full understanding of their pathophysiology must include an explanation for this important phenomenon, which plays a considerable role in modifying their phenotypic expression.
The complex topic of hemoglobin switching has been the subject of several extensive reviews.7,9,10,34 b-Globin synthesis commences early during fetal life, at approximately 8 to 10 weeks’ gestation. Subsequently, it continues at a low level, approximately 10 percent of the total non-a-globin chain production, up to about 36 weeks’ gestation, after which it is considerably augmented. At the same time, g-globin chain synthesis starts to decline so that at birth there are approximately equal amounts of g- and b-globin chains produced. Over the first year of life there is a gradual decline in g-chain synthesis, and by the end of the first year this amounts to less than 1 percent of the total non-a-globin chain output. In adults the small amount of hemoglobin F is confined to an erythrocyte population called F cells.
It is still not clear how this series of developmental switches is regulated. It is not organ specific but is synchronized throughout the developing hematopoietic tissues. Although environmental factors may be involved, the bulk of experimental evidence suggests that there is some form of “time clock” built into the hematopoietic stem cell. At the chromosomal level it appears that regulation occurs in a complex manner involving both developmental stage–specific trans-activating factors and the relative proximity of the different genes of the b-globin gene cluster to LAR. The elements involved in the stage-specific regulation of the human globin genes have not yet been identified.
Fetal hemoglobin synthesis may be reactivated at a low level in states of hematopoietic stress and occurs at higher levels in certain hematologic malignancies, notably juvenile myeloid leukemia. However, it is only in the hemoglobinopathies that high levels of hemoglobin F production are seen with any consistency in adult life.
MOLECULAR BASIS OF THE THALASSEMIAS
Once it became possible to clone and sequence the globin genes from patients with many different forms of thalassemia, it became clear that a wide spectrum of mutations underlie these conditions. A picture of remarkable heterogeneity has emerged. Indeed, it seems likely that the study of these disorders has already given us a fairly complete account of the repertoire of the types of molecular lesions that underlie human single-gene disorders. For more extensive coverage, the reader is referred to a number of recent monographs and reviews.9,10,35,36
b THALASSEMIA
b Thalassemia is extremely heterogeneous at the molecular level35,36; nearly 180 different mutations have been found in association with this phenotype. Broadly, they fall into deletions of the b-globin gene and nondeletional mutations that may affect the transcription, processing, or translation of b-globin messenger (Table 46-2 and Fig. 46-3). Each major population group has a different set of b thalassemia mutations, usually consisting of two or three that make up the bulk combined with large numbers of rare ones. Because of this pattern of distribution, only about 20 alleles account for the majority of all b thalassemia determinants.

TABLE 46-2 MOLECULAR PATHOLOGY OF THE b THALASSEMIAS

FIGURE 46-3 The classes of mutations that underlie b thalassemia. PR, promoter; C, CAP site; I, initiation site; FS, frameshift; NS, nonsense mutation; SPL, splicing mutation; POLY A, polyA addition site mutation.

Gene Deletions At least seventeen different deletions affecting only the b genes have now been described.10,36,37 With one exception, these are rare and appear to be isolated, single events; the 619-bp deletion at the 3′ end of b gene is more common,38 but even that is restricted to the Sind and Gujarati populations of Pakistan and India, where it accounts for approximately 50 percent b-thalassemia alleles.39 The Indian 619-bp deletion removes the 3′ end of the b gene but leaves the 5′ end intact, while many of the other deletions remove the 5′ end of the gene and leave the d gene intact.40,41,42,43,44 and 45 Homozygotes for these deletions have bo thalassemia. Heterozygotes for the Indian deletion have raised hemoglobin A2 and F levels identical to those seen in heterozygotes for the other common forms of b thalassemia. It is interesting, however, that heterozygotes for the other deletions all have unusually high hemoglobin A2 levels. The increased d-chain production results from increased d-gene transcription in cis to the deletion, possibly as a result of reduced competition from the deleted 5′ b gene for transcription factors.
Transcriptional Mutations Several different base substitutions have been found that involve the conserved sequences upstream from the b-globin gene.35,36 In every case the phenotype is b+ thalassemia, although there is considerable variability in the clinical severity associated with different mutations of this type. Several of them, at positions –88 and –87 relative to the mRNA CAP site, for example,46,47 are close to the CCAAT box, while the others lie within the ATA box homology.48,49,50 and 51
Some of the mutations upstream from the b-globin gene are associated with even more subtle alterations in phenotype. For example, a C®T substitution at position –101, which involves one of the upstream promotor elements, is associated with “silent” b thalassemia, that is, a completely normal phenotype that can be identified only by its interaction with more severe forms of b thalassemia in compound heterozygotes.52 A single example of an A ® C substitution at the CAP site (+1) was described in an Asian Indian who, despite being homozygous for the mutation, appeared to have the phenotype of the b-thalassemia trait.53
The upstream regulatory mutations confirm the importance of the conserved sequences in this region in their role as regulators of the transcription of the b-globin genes and provide the basis for some of the mildest forms of b thalassemia, particularly those in African populations.
RNA-Processing Mutations One of the surprises about b-thalassemia has been the remarkable diversity of the single-base mutations that can interfere with the intranuclear processing of mRNA.
The boundaries of exons and introns are marked by invariant dinucleotides, GT at the 5′ (donor) and AG at the 3′ (receptor) sites. Single-base changes that involve either of these splice junctions totally abolish normal RNA splicing and result in the phenotype of b0 thalassemia.36,47,50,54,55,56,57 and 58
Surrounding the invariant dinucleotides at the splice junctions are highly conserved sequences that are involved in mRNA processing. Different varieties of b thalassemia involve single-base substitutions within the consensus sequence of the IVS-1 donor site.36,47,50,54,55,59,60 and 61 These mutations are of particular interest because of the remarkable variability in their associated phenotypes. For example, substitution of the G in position 5 of IVS-1 by C or T results in severe b+ thalassemia.47 On the other hand, a T ® C change at position 6, found commonly in the Mediterranean region,62 results in a very mild form of b+ thalassemia. The G ® C change at position 5 has also been found in Melanesia and appears to be the most common cause of b thalassemia in Papua New Guinea.63
RNA processing is also affected by mutations that create new splice sites within either introns or exons. Again, these lesions are remarkably variable in their phenotypic effect, depending on the degree to which the new site is utilized compared with the normal splice site. For example, the G ® A substitution at position 110 of IVS-1, which is one of the most common forms of b thalassemia in the Mediterranean region, leads to only about 10 percent splicing at the normal site and hence results in a phenotype of severe b+ thalassemia.64,65 Similarly, a mutation that produces a new acceptor site at position 116 in IVS-1 results in little or no b-globin mRNA production and the phenotype of bo thalassemia.66 Several mutations have been described that generate new donor sites within IVS-2 of the b-globin gene.36,47,60
Another interesting mechanism for abnormal splicing is the activation of donor sites within exons (Fig. 46-4). For example, within exon 1 there is a cryptic donor site in the region of codons 24 through 27. This site contains a GT dinucleotide; an adjacent substitution that alters it so that it more closely resembles the consensus donor splice site results in its activation, even though the normal site is active. Several mutations in this region can activate this site so that it is utilized during RNA processing, with the production of abnormal mRNAs.66,67,68,69 and 70 Three of them, A ® G in codon 19, G ® A in codon 26, and G ® T in codon 27, result in both reduced production of b-globin mRNA and an amino acid substitution, so that the mRNA that is spliced normally is translated into protein. The abnormal hemoglobins produced are hemoglobins Malay, E, and Knossos, respectively, all of which are associated with a b-thalassemia phenotype, presumably due to the reduced overall output of normal mRNA. A variety of other cryptic splice mutations within introns and exons have been described.35,36

FIGURE 46-4 The activation of cryptic splice sites in exon 1 as the cause of b+ thalassemia, Hb E, and Hb Knossos. The similarities between the 5′ splice region of intron 1 and the cryptic splice region in exon 1 are shown in capitals.

Another class of processing mutations involves the polyadenylation signal site AAUAAA in the 3′ untranslated region of b-globin mRNA.71,72 and 73 For example, a T ® C substitution in this sequence leads to the transcription of only a tenth of the normal amount of b-globin mRNA and hence in the phenotype of severe b+ thalassemia.71
Mutations That Cause Abnormal Translation of Messenger RNA Base substitutions that change an amino acid codon into a chain termination codon, nonsense mutations, prevent translation of the mRNA and result in bo thalassemia. Many substitutions of this type have been described,36 a codon 17 mutation being common in Southeast Asia74,75 and a codon 39 mutation occurring at a high frequency in the Mediterranean region.76,77
The insertion or deletion of one, two, or four nucleotides in the coding region of b-globin gene disrupts the normal reading frame and results, on translation of the mRNA, in the addition of anomalous amino acids until a termination codon is reached in the new reading frame. Several frameshift mutations of this type have been described,36 and two, the insertion of one nucleotide between codons 8 and 9 and a deletion of four nucleotides in codons 41 and 42, are common in Asian Indians,55 with the latter also being found frequently in different populations in Southeast Asia.75
Dominantly Inherited b Thalassemia Over the past 20 years there have been sporadic reports of families in which a picture indistinguishable from moderately severe b thalassemia has segregated in Mendelian dominant fashion.78,79 Because this condition is often characterized by the presence of inclusion bodies in the red cell precursors, it has been called inclusion body b thalassemia, although, since all severe forms of b thalassemia have inclusions in the red cell precursors, the term dominantly inherited b thalassemia is preferred.80 Sequence analysis has shown that these conditions are heterogeneous at the molecular level but that many of them involve mutations of exon 3 of the b-globin gene (Table 46-3). These include frameshifts, premature chain termination mutations, and complex rearrangements that lead to the synthesis of truncated or elongated and highly unstable b-globin gene products.18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,
57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88 and 89 The most common mutation of this type is a GAA ® TAA change at codon 121 that leads to the synthesis of a truncated b-globin chain.85 Although it is unusual to demonstrate an abnormal b-chain product from loci affected by mutations of this type, many of these conditions have been designated as hemoglobin variants (see Table 46-3). As will be described in greater detail later, these b-globin products are unable to form a viable b chain and precipitate together with excess a chains in the marrow.

TABLE 46-3 SOME MOLECULAR FORMS OF DOMINANT b THALASSEMIA AND STRUCTURAL VARIANTS ASSOCIATED WITH A b THALASSEMIA PHENOTYPE

Unstable b-Globin Variants Some b-globin chain variants, although highly unstable, are capable of forming a viable tetramer. The resulting unstable hemoglobins may be precipitated in the red cell precursors or in the blood and hence give rise to a spectrum of conditions ranging from dominantly inherited b thalassemia to a hemolytic anemia similar to those associated with other unstable hemoglobins. The first of these to be described, hemoglobin Indianapolis,90 had its structure characterized by DNA analysis carried out on stored autopsy material; the original description turned out to be incorrect (see Table 46-3).91
Silent b Thalassemia It is now clear that there are a number of extremely mild b thalassemia alleles that are either silent or almost unidentifiable in heterozygotes (see Table 46-2). As mentioned earlier, some of these are in the region of the promoter boxes of the b-globin gene, but others involve the CAP sites or the 5′ or 3′ untranslated regions.35,36 These alleles are usually identified by the finding of a form of b thalassemia intermedia in which one parent has a typical thalassemia trait while the other appears to be normal but, in fact, is a carrier of one of these mild b-thalassemia alleles.
b-Thalassemia Mutations Unlinked to the b-Globin Gene Cluster Although several family studies have suggested that there may be mutations that result in the phenotype of b thalassemia that do not segregate with the b-globin genes,92 their molecular basis has not yet been determined. The evidence for the existence of novel mutations of this type has been reviewed recently.7,35
Variant Forms of b Thalassemia There are several forms of b thalassemia in which the level of hemoglobin A2 is normal in heterozygotes.10 In some cases these are due to “silent” b thalassemia alleles, while others reflect the coinheritance of b and d thalassemia.
db THALASSEMIA
The (db) thalassemias are classified into the (db)+ and (db)o thalassemias (Table 46-4). The (db)o thalassemias are further divided into those in which both the d- and b-globin genes are deleted, (db)o thalassemia, and those in which the Gg, d, and b genes are deleted, (Agdb)o thalassemia. Because many different deletion forms of db thalassemia have been described, they are further classified according to the country in which they were first identified (Table 46-4).

TABLE 46-4 THE db THALASSEMIAS

(db)o and (Agdb)o Thalassemia Nearly all these conditions result from deletions that involve varying lengths of the b-globin gene cluster. Many different varieties have been described in different populations (see Table 46-4), although their heterozygous and homozygous phenotypes are very similar.7,35,36,93 Rare forms of these conditions are due to more complex gene rearrangements. For example, one form of (Agdb)o thalassemia, found in Indian populations, is not due to a simple linear deletion, but, rather, it results from a complex rearrangement with two deletions, one affecting the Ag gene and the other the d and b genes; the intervening region is intact but inverted.94 Some of these conditions are illustrated in Fig. 46-5.

FIGURE 46-5 The various deletions that are responsible for the b and db thalassemias and hereditary persistence of fetal hemoglobin.

(db)+ Thalassemia The (db)+ thalassemias are usually associated with the production of structural hemoglobin variants called Lepore.7,9,10 Hemoglobin Lepore contains normal a chains and non-a chains that consist of the first 50 to 80 amino acid residues of the d chains and the last 60 to 90 residues of the normal C-terminal amino acid sequence of the b chains. Thus, the Lepore non-a chain is a db fusion chain. Several different varieties of hemoglobin Lepore have been described—Washington-Boston, Baltimore, and Hollandia—in which the transition from d to b sequences occurs at different points.7,10 The fusion chains have probably arisen by nonhomologous crossing over between part of the d locus on one chromosome and part of the b locus on the complementary chromosome (Fig. 46-6). This event results from misalignment of chromosome pairing during meiosis so that a d-chain gene pairs with a b-chain gene instead of with its homologous partner.95 As shown in Fig. 46-6, such a mechanism should give rise to two abnormal chromosomes: the first, the Lepore chromosome, will have no normal d or b loci but simply a db fusion gene. On the opposite of the homologous pairs of chromosomes there should be an anti-Lepore (bd) fusion gene together with normal d and b loci. A variety of anti-Lepore–like hemoglobins have been discovered, including hemoglobins Miyada, P-Congo, Lincoln Park, and P-Nilotic.9,10 All the hemoglobin Lepore disorders are characterized by a severe form of db thalassemia; the output of the g-globin genes on the chromosome with the db fusion gene is not increased sufficiently to compensate for the low output of the db fusion product. The reduced rate of production of the db fusion chains of hemoglobin Lepore presumably reflects the fact that its genetic determinant has the d gene promoter region, which is structurally different from the b-globin gene promoter and is associated with a reduced rate of transcription of its gene product.

FIGURE 46-6 The mechanisms of the production of the Lepore and anti-Lepore hemoglobins and Hb Kenya.

db Thalassemia–like Disorders Due to Two Mutations in the b-Globin Gene Cluster A heterogeneous group of nondeletion db thalassemias has been described, most of which result from two mutations in the egdb-globin gene cluster (see Table 46-4). Strictly speaking, they are not all db thalassemias, but because their phenotypes resemble the deletion forms of (db)o thalassemia, they often appear in the literature under this title. In the Sardinian form of db thalassemia, the b-globin gene has the common Mediterranean codon 39 nonsense mutation that leads to an absence of b-globin synthesis. However, there is relatively high expression of the Ag gene in cis, which gives this condition the phenotype db thalassemia; this is because there is a point mutation at position –196 upstream from the Ag gene (see “Hereditary Persistence of Fetal Hemoglobin”). The phenotypic picture, in which heterozygotes have 15 to 20 percent hemoglobin F and normal levels of hemoglobin A2, is identical to that of db thalassemia.96 Another condition that has the phenotype db thalassemia, with over 20 percent hemoglobin F in heterozygotes, has been described in a Chinese patient in whom defective b-globin–chain synthesis appears to be due to an A ® G change in the ATA sequence in the promoter region of the b-globin gene.97 However, the increased g-chain synthesis, which appears to involve both Gg and Ag cis to this mutation, remains unexplained. A disorder that was originally called db thalassemia has been described in the Corfu population.98,99 Again, this results from two mutations in the b-globin gene cluster. First, there is a 7201-bp deletion that starts in the d-globin gene, IVS-2, position 818–822, and extends upstream to a 5′ breakpoint located 1719 to 1722 bp 3′ to the yb-gene termination codon. In addition there is a G ® A mutation at position 5 in the donor site consensus region of IVS-1 of the b-globin gene. The output from this chromosome consists of relatively high levels of g chains with very low levels of b chains. This condition resembles db thalassemia in the homozygous state, in which there is almost 100 percent hemoglobin F, with traces of hemoglobin A but no hemoglobin A2; heterozygotes have only slightly elevated levels of hemoglobin F, and the phenotype is similar to “normal A2 b thalassemia.”
egdb THALASSEMIA
These rare conditions100,101,102,103,104,105 and 106 result from long deletions that begin upstream from the b-gene complex 55 kb or more 5′ to the e gene and terminate within the cluster (see Fig. 46-5). In two cases, designated Dutch103,104 and English,105 the deletions leave the b-globin gene intact, but no b-chain production occurs even though the gene is expressed in heterologous systems.
The molecular basis for the inactivation of the b-globin gene cis to these deletions was clarified by the discovery of the LCR about 50 kb upstream from the egdb-globin gene cluster (see “Regulation of Globin Gene Clusters”). The removal of this critical regulatory region seems to completely inactivate the downstream globin gene complex. The Hispanic form of egdb thalassemia106 results from a deletion that includes most of the LAR, including four of the five DNase-1–hypersensitive sites. These lesions appear to close down the chromatin domain that is usually open in erythroid tissues. They also delay the replication of the b-globin genes in the cell cycle. Thus, although they are rare, they have been of considerable importance, since it was the analysis of the Dutch deletion that first pointed to the possibility of there being a major control region upstream from the b-like–globin gene cluster and ultimately led to the discovery of the b-globin LCR.
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
This heterogeneous group of conditions produces phenotypes very similar to those of the db thalassemias except that defective b-chain production appears to be almost, although in some forms not completely, compensated by persistent g-chain production. These conditions are best classified into deletion and nondeletion forms (Table 46-5). Although in the past it was customary to further classify them into pancellular and heterocellular varieties, depending on the intercellular distribution of fetal hemoglobin, this now appears to bear little relevance to their molecular basis and probably relates more to the particular level of fetal hemoglobin and to the way in which its cellular distribution is determined.7

TABLE 46-5 HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

The deletion forms of HPFH are heterogeneous (see Fig. 46-5). The two African varieties are due to extensive deletions of similar length (<70 kb) but with staggered ends, differing phenotypically only in the proportions of Gg and Ag chains produced.107 Another type of HPFH results from misalignment during crossing over between the Ag and b-globin genes, resulting in the production of Agb fusion genes (see Fig. 46-6). The latter give rise to gb fusion products that combine with a chains to form the hemoglobin variant called hemoglobin Kenya.108,109 This is associated with an increased output of hemoglobin F, although at a lower level than the deletion forms of HPFH described above. So far, it has not been possible to develop a theory that provides an adequate explanation for the phenotypic differences between db thalassemia and the deletion forms of HPFH. As mentioned earlier, they form a continuum of conditions that differ only in the relative output of g chains directed by the chromosome carrying a particular deletion. Based on the observation that as a group the HPFH deletions tend to extend farther upstream than do those that produce db thalassemia, attempts have been made to define putative regulatory regions in the b-globin gene cluster that may or may not be involved in their particular deletions.98,110 For example, the 5′ ends of the HPFH and db-thalassemia deletions that lie closest together have been analyzed in detail. It has been found that the two deletions end in a pair of Alu 1 repeats 5′ to the d gene.98,110 The HPFH deletion ends in the 5′ Alu 1 repeat of the bipolar pair, while the db-thalassemia deletion ends in the 3′ Alu 1 repeat. Thus, the two deletions have endpoints that are within 500 nucleotides of each other; the larger deletion causes a significantly higher output of g chains than does the smaller one. Therefore, unless the two different phenotypes result from differences in the DNA sequences at the 3′ end of the deletions, the 5′ Alu 1 repeat and the nonrepetitive DNA connecting it to the 3′ Alu repeat must be considered to play an important regulatory role. Alternatively, it has been pointed out that the deletions that cause HPFH are situated at least 52 to 57 kb from the 3′ extremity of the b-globin genes, while most of those that cause db thalassemia are shorter and are located no more than 5 to 10 kb from the b gene. Accordingly, it has been suggested107 that the nature of the DNA brought into the vicinity of the g genes by these deletions, possibly enhancer sequences, may be an important factor in determining the phenotype. On the other hand, at least three (Agdb)o thalassemias have been shown to have different 3′ sequences, yet their phenotypes are essentially the same. In fact, the phenotypes of several deletions involving this gene cluster are incompatible with this hypothesis.
The nondeletion determinants of HPFH can be classified into those that map within the b-globin gene cluster and those that segregate independently. The former are subdivided into Ggb+ and Agb+ varieties, indicating that there is persistent Gg- or Ag-chain synthesis in association with b-globin production directed by the b gene cis (on the same chromosome) to the HPFH determinant. Analysis of the overexpressed g genes has revealed in each case a single-base substitution in the region immediately upstream from the transcription start site.36,111,112,113 and 114 The clustering of these substitutions and the lack of similar changes in normal g genes suggest that they are responsible for the persistent hemoglobin F production (Fig. 46-7). It seems likely that this region of DNA is involved in the binding of trans-acting proteins involved in the normal developmental repression of g-gene expression, either by decreasing the affinity for an inhibitory factor normally present in adult life or by increasing the affinity for a factor for promoting gene expression. The most common of these conditions are Greek Agb+ HPFH and a form of Ggb+ HPFH which has been found in several different African populations. It is becoming apparent that, if these upstream point mutations that are associated with persistent g-chain production occur on the same chromosome as b-globin genes that carry bo thalassemia mutations, the clinical phenotype is converted from HPFH to db thalassemia.

FIGURE 46-7 Some of the upstream point mutations associated with hereditary persistence of fetal hemoglobin.

There are other nondeletional forms of HPFH that in some cases have been related to small structural changes in the b-globin gene cluster (see Table 46-4). Although strictly speaking not a true form of HPFH, since even in homozygotes it may not be associated with increased levels of hemoglobin F, the T ® C polymorphism at position –158 to the Gg-globin gene115 may, under conditions of erythropoietic stress, be associated with an increased output of hemoglobin F.
There are other forms of HPFH characterized by the persistence of low levels of fetal hemoglobin production distributed in a heterocellular manner. In all populations studied, a small proportion of individuals have an increased amount of hemoglobin F and F cells, that is, red cells that can be detected when blood films are treated with antibodies against hemoglobin F. Although this condition was originally called the Swiss form of HPFH because it was first recognized in Swiss army recruits,116 it has now been observed in every racial group. There is some evidence that at least one genetic determinant is responsible for determining the number of F cells is X-linked, and a putative locus has been located at Xp22.2.117,118 However, it is clear that not all forms of hereditary persistence of low levels of hemoglobin F are encoded by the X chromosome.119,120,121 and 122 In studies of a large pedigree in which a form of HPFH segregated independently from b thalassemia, the genetic determinant has been localized to chromosome 6q23.123 However, further studies have indicated that similar forms of HPFH are not linked to chromosome 6.124 It seems likely that these different forms of HPFH that are unlinked to the b-globin gene cluster reflect mutations of transcription factors that are involved in the switch from feteal to adult hemoglobin production. The importance of these conditions lies in the fact that, when they are inherited together with the sickle-cell or b-thalassemia genes, they may increase the output of hemoglobin F to such an extent that they modify the phenotype of the associated disorders.
d THALASSEMIA
Several point mutations and deletions that reduce d-globin synthesis have been described. They are summarized in Fig. 46-8.

FIGURE 46-8 The molecular basis of some of the d thalassemias (references to original descriptions in Ref. 36).

a THALASSEMIA
The different classes of a thalassemia are summarized in Table 46-6. The a-globin gene haplotype can be written aa, indicating the a2 and a1 genes, respectively. A normal individual has the genotype aa/aa. A deletion involving one (–a) or both (––) a genes can be further classified on the basis of its size, written as a superscript; thus, –a3.7 indicates a deletion of 3.7 kb including one a gene. When the sizes of the deletions have not yet been established, a superscript describing their geographic or family origin is useful; thus, – –MED describes a deletion of both a genes first identified in individuals of Mediterranean origin. In those thalassemia haplotypes where both genes are intact, that is, nondeletion lesions, the nomenclature aTa is given, the superscript T indicating that this gene is thalassemic. However, when the precise molecular defect is known, as in hemoglobin Constant Spring, for example, aTa can be replaced by the more informative aCSa. The molecular pathology and population genetics of the a thalassemias have been the subject of several extensive reviews.7,36,125

TABLE 46-6 CLASSES OF MUTATIONS THAT CAUSE a THALASSEMIA+

a0 Thalassemia To date, 14 deletions that involve both a genes, and therefore abolish a-chain production from the affected chromosome, have been described (Fig. 46-9).7,36 Several of the 3′ breakpoints fall within a 6- to 8-kb region at the 3′ end of the a-globin complex, suggesting that this may represent a breakpoint cluster region with a high level of recombination.126 In at least five of the deletions, the 5′ breakpoints also appear to cluster. This gives rise to a situation in which the 5′ breakpoints are located approximately the same distance apart and in the same order along a chromosome as their respective 3′ breakpoints. It is possible that such staggered deletions may have arisen from illegitimate recombination events that delete an integral number of chromatin loops as they pass through their nuclear attachment points during replication, a mechanism that has also been suggested to underlie some of the deletion forms of HPFH.127 One of these deletions (– –MED) involves a more complex rearrangement that introduces a new piece of DNA bridging the two breakpoints in the a-gene cluster. This new sequence originates upstream from the a cluster and appears to have been replicated into the junction in a manner that suggests that the upstream segment of DNA also lies at the base of a replication loop. At least some of these deletions seem to have arisen by recombination events between Alu repeat sequences.

FIGURE 46-9 The deletions of the a globin gene cluster that are responsible for ao thalassemia. Deletions: MC = initials of patient; CAL = initials of patient; THAI = Thai; FIL = Filipino; CI = Conway Islands; BRIT = UK; SA = South Africa; MED = Mediterranean; SEA = Southeast Asian; SPAN = Spanish.

Several other mechanisms for the generation of ao thalassemia have been identified.7,125 In some cases this condition results from a terminal truncation of the short arm of chromosome 16 to a site 50 kb distal to the a-globin genes.128 It is interesting to note that the telomeric consensus sequence (TTAGGGG)n has been added directly to the site of the break. Since this mutation is stably inherited, it appears that telomeric DNA alone is sufficient to stabilize the broken chromosome end. This observation raises the possibility that other genetic diseases may result from chromosomal truncations.
Several deletions have now been identified that appear to downregulate the a-globin genes by removal of the a-globin locus control region (HS40).7,129,130 In each case the a-globin genes have been left intact, although in one the 3′ breakpoint is found between the x2 and yx1 genes, thus removing the x2 gene.129 It appears that these deletions completely inactivate the a-globin gene complex, just as deletions of the b-globin LAR inactivate the entire b-gene complex. So far, such deletions have not been observed in the homozygous state, presumably because they would be lethal.
a+ Thalassemia Gene Deletions The most common forms of a+ thalassemia (a3.7 and –a4.2) involve the deletion of one or the other of the duplicated a-globin genes (Fig. 46-10).

FIGURE 46-10 The mechanisms for the production of the common deletion forms of a+ thalassemia: (a) the normal a-globin gene cluster showing the homology boxes X, Y, and Z; (b) the rightward crossover through the Z bones, giving rise to the 3.7-kb deletion and a chromosome with 3 a-globin genes; (c) the leftward crossover through the Z boxes, giving rise to a 4.2-kb deletion and a chromosome containing 3 a genes.

Each a gene is located within a region of homology approximately 4 kb long, interrupted by two nonhomologous regions. It is thought that the homologous regions have resulted from an ancient duplication event and that subsequently they were subdivided, presumably by insertions and deletions, to give three homologous subsegments referred to as X, Y, and Z (see Fig. 46-10). The duplicated Z boxes are 3.7 kb apart, and the X boxes are 4.2 kb apart. Misalignment and reciprocal crossover between these segments at meiosis can give rise to chromosomes with either single (–a) or triplicated (aaa) a-globin genes. Such an occurrence between homologous Z boxes deletes 3.7 kb of DNA (rightward deletion), while a similar crossover between the two X blocks deletes 4.2 kb of DNA (leftward deletion –a4.2).131 The corresponding triplicated a-gene arrangements are referred to as aaaanti-3.7 and aanti-4.2.132,133 and 134 More detailed analysis of these crossover events indicates that they occur more commonly in the Z box, and at least three different –a3.7 deletions have been found, depending on exactly where the crossover has taken place.135 These are designated –a3.7I, –a3.7II, and –a3.7III, respectively. Other, rarer deletions of a single a gene have been observed.7,125
Nondeletion a Thalassemia Since the expression of the a2 gene is two to three times greater than that of the a1 gene, it is not surprising that most of the nondeletion mutants discovered to date affect predominantly the expression of the a2 gene; presumably this is ascertainment bias because of the greater phenotypic effect of these lesions. It is also possible that they have come under greater selective pressure.
Like the b-thalassemia mutations, a-thalassemia mutations7,36,125 can be classified according to the level of gene expression they affect (see Table 46-5). Several processing mutations have been identified. For example, a pentanucleotide deletion includes the 5′ splice site of IVS-1 of the a2-globin gene. This involves the invariant GT donor splicing sequence and thus completely inactivates the a2 gene.136 A second mutant of this type, found commonly in the Middle East, involves the poly-A addition signal site (AATAAA ® AATAAG) and downregulates the a2 gene by interfering with 3′ end processing.137,138
A second group of nondeletion a thalassemias result from mutations that interfere with the translation of mRNA.7,36,139,140,141 and 142 In one case, for example, the initiation codon is inactivated by a T ® C transition,139 and, in another, efficiency of initiation is reduced by a dinucleotide deletion in the consensus sequence around the start signal.142 Five mutations that affect termination of translation and give rise to elongated a chains have been identified: hemoglobins Constant Spring, Icaria, Koya Dora, Seal Rock, and Pakse.7,36,143 Each specifically changes the termination codon TAA so that an amino acid is inserted instead of the chain terminating (Fig. 46-11). This is followed by read-through of mRNA that is not normally translated until another “in-phase” stop codon is reached. Thus, each of these variants has an elongated a chain. It seems likely that the “read-through” of a-globin mRNA that is usually not utilized somehow reduces its stability. There are several nonsense mutations, one in exon 3 of the a2-globin gene, for example.144 Finally, there are several mutations that cause a thalassemia by producing highly unstable a-globin chains; they include hemoglobins Quong Sze,145 Suan Doc,146 Petah Tikvah,147 and Evanston.148 A full list of nondeletion a thalassemia alleles is given in Ref.7 and 36.

FIGURE 46-11 Point mutations in the a globin gene termination codon.

The Molecular Pathology of the a-Thalassemia–Mental Retardation Syndrome The first descriptions of noninherited forms a thalassemia associated with mental retardation suggested that the lesions involving the a-globin gene locus might be acquired in the paternal germ cells and that their molecular pathology might help elucidate the associated developmental changes.149 It is now clear that there are two separate syndromes of this type. In one group of patients there are long deletions involving the a-globin gene cluster and removing at least one megabase.150 It appears that this condition can arise in several ways, including unbalanced translocation involving chromosome 16, truncation of the tip of chromosome 16, and the loss of the a-globin gene cluster and parts of its flanking regions by other mechanisms. These findings localize a region of about 1.7 Mb in band 16p13.3 proximal to the a-globin genes as being involved in mental handicap.7,150
The second group is characterized by defective a-globin synthesis associated with severe mental retardation and a relatively homogeneous pattern of dysmorphology.151 Extensive structural studies have shown no abnormalities of the a-globin genes, the activity of which appears to be reduced in both cis and trans. These chromosomes direct the synthesis of normal amounts of a globin in mouse erythroleukemia cells, suggesting that the a thalassemia is due to a deficiency of a trans-activating factor involved in the regulation of the a-globin genes. This condition is encoded by a locus on the short arm of the X chromosome.152 The gene involved has now been identified. It is XH2, a DNA helicase with many features of a DNA-binding protein.153 Many different mutations of this gene have already been identified in different families with the ATR-X syndrome.154 The murine homolog of this gene is widely expressed during early development. It is currently believed that the XH2 product is a ubiquitous transcription factor that is involved in early development, particularly of the urogenital system and brain, which also acts as a transcription factor for the a-globin genes.
Interactions of a-Thalassemia Haplotypes Many a-thalassemia haplotypes have been described, and there are potentially over 500 interactions!7,125 Phenotypically, these result in one of four broad categories: normal, conditions in which there are mild hematologic changes but no clinical abnormality, hemoglobin H disease, and the hemoglobin Bart’s hydrops fetalis syndrome. The heterozygous states for deletion or nondeletion forms of a+ thalassemia either cause extremely mild hematologic abnormalities or are completely silent. In populations where a thalassemia is common, the homozygous state for a+ thalassemia (–a/–a) can produce a hematologic phenotype identical to that of the heterozygous state for ao thalassemia (– –/aa), that is, mild anemia with reduced MCH and MCV values.
Hemoglobin H disease usually results from the compound heterozygous state for ao thalassemia and either deletion or nondeletion a+ thalassemia. It occurs most frequently in Southeast Asia (– –SEA/–a3.7) and the Mediterranean region (usually – –MED/–a3.7). Hemoglobin H disease may also result from the homozygous state for nondeletion mutants affecting the a2 gene (aaT Saudi/aaT Saudi).7,125
The hemoglobin Bart’s hydrops fetalis syndrome usually results from the homozygous state for ao thalassemia, most commonly – –SEA/– –SEA or – –MED/– –MED. There have been a few reports of infants with this syndrome who synthesized very low levels of a chains at birth. Gene-mapping studies suggest that these cases result from the interaction of ao thalassemia with nondeletion mutations (aaT).155 Recent studies suggest that the latter lead to the production of highly unstable a-chain variants.156,157
PATHOPHYSIOLOGY
It is possible to relate almost all the pathophysiologic features of the thalassemias to a primary imbalance of globin-chain synthesis. It is this phenomenon that makes them fundamentally different from all the other genetic and acquired disorders of hemoglobin production and that to a large extent explains their extreme severity in the homozygous or compound heterozygous states (Fig. 46-12).

FIGURE 46-12 The pathophysiology of b thalassemia.

IMBALANCED GLOBIN-CHAIN SYNTHESIS
Measurements of in vitro globin-chain synthesis in the blood or marrow of patients with different types of thalassemia,158,159 together with family studies that enable the action of the thalassemia genes to be examined in patients who have also inherited a- or b-globin structural variants,8 provide a clear picture of the action of the thalassemia determinants.7 In homozygous b thalassemia, b-Globin synthesis is either absent or markedly reduced. This results in the production of an excess of a-globin chains. a-Globin chains are incapable of forming a viable hemoglobin tetramer and hence precipitate in red cell precursors. The resulting inclusion bodies can be demonstrated by both light161 and electron microscopy.161 and 162 In the marrow, precipitation can be seen in the earliest hemoglobinized precursors and through the erythroid maturation pathway.163 These large inclusions are responsible for the intramedullary destruction of red cell precursors and hence for the ineffective erythropoiesis that characterizes all the b thalassemias. It has been calculated that a large proportion of the developing erythroblasts are destroyed within the marrow in severe cases.164 Any red cells that are released are prematurely destroyed by mechanisms that are considered below. b-Thalassemia heterozygotes also have imbalanced globin-chain synthesis, but in this case the magnitude of the excess of a chains is much less and presumably can be dealt with successfully by the proteolytic enzymes of the red cell precursors.165 Notwithstanding, there is a mild degree of ineffective erythropoiesis.
It appears that there are two major routes to damage of the red cell membrane by the globin-chain precipitation process: the generation of hemichromes from excess a chains with subsequent structural damage to the red cell membrane, and similar damage mediated through the degradation products of excess a chains.166,167 and 168 Membrane-bound hemichromes create a copolymer, which promotes clustering of band 3 in the membrane, first observed in sickle cell erythrocytes and later in the red cells of b thalassemics. It seems likely that these clusters are opsonized with autologous immunoglobulin G and complement, after which the cells are removed by macrophages. The products of degradation of free a chains—that is, globin, heme, hemin (oxidized heme), and free iron—also play a role in damaging red cell membranes. Excess globin chains bind to different membrane proteins and alter their structure and function. Excess iron, by generating oxygen free radicals, damages several red cell membrane components, including lipids and protein, as well as intracellular organelles. Heme and its products can catalyze the formation of a variety of reactive oxygen species which can produce damage to the red cell membrane. In b thalassemia this leads to a relatively rigid, underhydrated red cell. Damage to the red cells may also be mediated during their passage through the spleen due to the presence of rigid inclusion bodies.
While most of b-thalassemia heterozygotes are asymptomatic and have a mild hypochromic anemia, there are more severe forms that are dominantly inherited. Many of these involve mutations in exon 3 of the b-globin gene. A comparison of the lengths of abnormal gene products due to nonsense or frameshift mutations in the b-globin gene have suggested a mechanism that explains why most heterozygous forms of b thalassemia are mild, while those due to exon 3 mutations are more severe.80,81 Nonsense or frameshift mutations that produce truncated b chains up to about 72 residues in length are usually associated with a mild phenotype in heterozygotes. It appears that mRNA containing stop or frameshift mutations in its 5′ regions may not be transported to the cytoplasm. However, many exon 3 mutations produce normal amounts of mRNA and long, truncated products. It has been suggested that the severe phenotypes associated with them reflect their heme-binding properties and stability. Those with only 72 residues or less cannot bind heme, while those truncated to residue 120 or longer should bind heme, since only helix H is missing. Furthermore, such heme-containing products should have a secondary structure and hence be less susceptible to proteolytic degradation. The lack of helix H, which would expose one of the hydrophobic patches of helix G and the hydrophobic patches of helices E and F, would tend to lead to aggregation of the truncated products. It was suggested, therefore, that the large inclusions in the red cell progenitors of these patients consist of aggregates of precipitated b-chain products together with excess a chains, a notion that has been shown to be correct.169 This explains the inclusion bodies in the red cell precursors and the marked degree of dyserythropoiesis that is observed in this interesting condition.
It is clear, therefore, that the anemia of b thalassemia has three major components. First and most important, there is ineffective erythropoiesis with intramedullary destruction of a variable proportion of the developing red cell precursors. Second, there is a hemolysis due to destruction of mature red cells containing a-chain inclusions. Third, because of the overall reduction in hemoglobin synthesis, the red cells are hypochromic and microcytic.
Because the primary defect in b thalassemia is in b-chain production, the synthesis of hemoglobins F and A2 should be unaffected. Fetal hemoglobin production in utero is normal, and it is only when the neonatal switch from g- to b-chain production occurs that the clinical manifestations of thalassemia first appear. However, fetal hemoglobin synthesis persists beyond the neonatal period in nearly all forms of b thalassemia (see Persistent Fetal Hemoglobin Production and Cellular Heterogeneity.) In b-thalassemia heterozygotes, there is an elevated level of hemoglobin A2. This appears to reflect not only a relative decrease in hemoglobin A due to defective b-chain synthesis but also an absolute increase in the output of d chains both cis and trans to the mutant b-globin gene.7
The consequences of excess non-a-chain production in the a thalassemias are quite different. Because a chains are shared by both fetal and adult hemoglobin, defective a-chain production is manifest in both fetal and adult life. In the fetus, it leads to excess g-chain production; in the adult, to an excess of b chains. Excess g chains form g4 homotetramers or hemoglobin Bart’s169; excess b chains form b4 homotetramers or hemoglobin H.169 The fact that g and b chains form homotetramers is the reason for the fundamental difference in the pathophysiology of a and b thalassemia. Because g4 and b4 tetramers are soluble, they do not precipitate to any significant degree in the marrow, and therefore the a thalassemias are not characterized by severe ineffective erythropoiesis. However, b4 tetramers precipitate as red cells age, with the formation of inclusion bodies.171 Thus, the anemia of the more severe forms of a thalassemia in the adult is due to a shortened survival of red cells consequent to their damage in the microvasculature of the spleen as a result of the presence of the inclusions. In addition, because of the defect in hemoglobin synthesis, the cells are hypochromic and microcytic. Hemoglobin Bart’s is more stable than hemoglobin H and does not form large inclusions.
It is now clear that, although, as is the case in b thalassemia, excess globin chains cause damage to the red cell membrane, the mechanisms are different in the two forms of the disease. As we saw, in the case of b thalassemia, excess a chains result in mechanical instability and oxidative damage to a variety of membrane proteins, notably protein 4.1. However, in a thalassemia the membranes are hyperstable and there is no evidence of oxidation or dysfunction of this protein. Furthermore, the state of red cell hydration is different in a thalassemia; accumulation of excess b chains results in increased hydration. These differences in the pathophysiology of membrane damage between a and b thalassemia are discussed in detail in Ref. 167 and 168.
There is another factor that exacerbates the tissue hypoxia of the anemia of the a thalassemias. Both hemoglobin Bart’s and hemoglobin H show no heme-heme interaction and have almost hyperbolic oxygen dissociation curves with very high oxygen affinities. Thus, they are not able to liberate oxygen at physiologic tissue tensions and are, in effect, useless as oxygen carriers.7,9,10
It follows, therefore, that infants with high levels of hemoglobin Bart’s have severe intrauterine hypoxia. This is the major basis for the clinical picture of homozygous ao thalassemia, which results in the stillbirth of hydropic infants late in pregnancy or at term. Oxygen deprivation is reflected by the grossly hydropic state of the infant, presumably due to an increase in capillary permeability, and by severe erythroblastosis. Deficient fetal oxygenation is probably responsible for the enormously hypertrophied placentas, and possibly for the associated developmental abnomalities, that occur with the severe forms of intrauterine a thalassemia.172
PERSISTENT FETAL HEMOGLOBIN PRODUCTION AND CELLULAR HETEROGENEITY
In children with severe thalassemia, there is an increased level of hemoglobin F that persists into childhood and later7,10; in the bo thalassemias, except for small amounts of hemoglobin A2, hemoglobin F is the only hemoglobin produced. Examination of the blood using staining methods that are specific for hemoglobin F show that it is heterogeneously distributed among the red cells.7 Persistent hemoglobin F production is not a major feature of the more severe forms of a thalassemia.
The mechanism of persistent g-chain synthesis in the thalassemias is still incompletely understood. Normal adults have small quantities of hemoglobin F that are heterogeneously distributed among the red cells; cells with demonstrable hemoglobin F are called F cells. It is clear that one important mechanism for high levels of hemoglobin F in the blood of patients with b thalassemia is cell selection.7,10,173,174 The major cause of ineffective erythropoiesis and shortened red cell survival in b thalassemia is the deleterious effect of excess a chains on erythroid maturation in the marrow and on the survival of red cells in the blood. It follows, therefore, that red cell precursors that produce g chains will be at a selective advantage; excess a chains combine with g chains to produce hemoglobin F, and therefore the magnitude of a-chain precipitation is less. Differential centrifugation experiments174 and in vivo labeling studies173 have shown that populations of red cells with relatively large amounts of hemoglobin F are more efficiently produced and survive longer in the blood. In fact, the blood of patients with homozygous b thalassemia shows remarkable cellular heterogeneity with respect to red cell survival; there are populations of cells that contain predominantly hemoglobin A that are destroyed very rapidly in the spleen and elsewhere, cells with a much longer survival that contain relatively more hemoglobin F, and populations of intermediate age and hemoglobin constitution.7,10,175
Whether cell selection of this type is the only mechanism for persistent g-chain production in b thalassemia is uncertain. It is possible that there is also an absolute increase in hemoglobin F production; this is certainly so in some milder forms of homozygous bo thalassemia, but in these cases there may be other genetic factors that are responsible for the relatively high level of g-chain synthesis (see below). However, biosynthesis studies indicate that marrow expansion and the selective survival of F-cell precursors and their progeny are the major factors in hemoglobin F production in hemoglobin E/b thalassemia.176
Since there is a reciprocal (see Fig. 46-9) relation between g- and d-chain synthesis, it follows that the red cells of b-thalassemia homozygotes that contain large amounts of hemoglobin F have relatively low levels of hemoglobin A2.7,10 Thus, the measured percent hemoglobin A2 in these individuals is the average of a very heterogeneous cell population. This probably accounts for the extreme variability in the levels of hemoglobin A2 found in homozygotes for this disorder. A further consequence of the persistence of hemoglobin F in b thalassemia is that the red cells have a high oxygen affinity.
CONSEQUENCES OF COMPENSATORY MECHANISMS FOR THE ANEMIA OF THALASSEMIA
The profound anemia of homozygous b thalassemia and the high oxygen affinity of the blood that is produced combine to cause severe tissue hypoxia. Because of the high oxygen affinity of hemoglobins Bart’s and H, a similar defect in tissue oxygenation occurs in the more severe forms of a thalassemia. The major response is erythropoietin production and expansion of the dyserythropoietic marrow. This in turn leads to deformities of the skull and face and porosity of the long bones.7 In extreme cases extramedullary hematopoietic tumors may develop. Apart from the production of severe skeletal deformities, marrow expansion may cause pathologic fractures and sinus and middle ear infection due to ineffective drainage.
Another important effect of the enormous expansion of the marrow mass is the diversion of calories required for normal development to the ineffective red cell precursors. Thus, patients severely affected by thalassemia show poor development and wasting. The massive turnover of erythroid precursors may result in secondary hyperuricemia and gout and severe folate deficiency.
The effects of gross intrauterine hypoxia in homozygous ao thalassemia have already been described. In the symptomatic forms of a thalassemia (e.g., hemoglobin H disease) that are compatible with survival into adult life, bone changes and other consequences of erythroid expansion are seen, although less commonly than in b thalassemia.
SPLENOMEGALY: DILUTIONAL ANEMIA
The constant exposure of the spleen to red cells with inclusions consisting of precipitated globin chains gives rise to the phenomenon of “work hypertrophy.” Progressive splenomegaly occurs in both a and b thalassemia and may exacerbate the anemia.7,10,177 A large spleen acts as a sump for red cells and may sequester a considerable proportion of the red cell mass. Furthermore, splenomegaly may also cause plasma volume expansion, a complication that may be exacerbated by massive expansion of the erythroid marrow. The combination of pooling of the red cells in the spleen together with plasma volume expansion may exacerbate the anemia in both a and b thalassemia. The same process may occur in an enlarged liver, particularly after splenectomy.
ABNORMAL IRON METABOLISM
b-Thalassemia homozygotes who are anemic manifest increased intestinal iron absorption that is related to the degree of expansion of the red cell precursor population; iron absorption is decreased by blood transfusion.10,177 Increased absorption causes a steady accumulation of iron, first in the Kupffer cells of the liver and the macrophages of the spleen and later in the parenchymal cells of the liver. Most patients homozygous for b thalassemia require regular blood transfusion, and thus transfusional siderosis adds to the iron accumulation. Iron accumulates in the endocrine glands,7,177,178,179 and 180 particularly in the parathyroids, pituitary, and pancreas, as well as in the liver, and, most important, in the myocardium.7,181 The latter leads to death either by involving the conducting tissues or by causing intractable cardiac failure. Other consequences of iron loading include diabetes, hypoparathyroidism, hypothyroidism, and abnormalities of hypopthalamic-pituitary function leading to growth retardation and hypogonadism.7,178,180
There is now much more accurate information about the levels of body iron, as reflected by hepatic iron, at which patients are at risk for serious complications of iron overload.182 These studies, which extrapolate data obtained from patients with genetic hemochromatosis, suggest that patients with hepatic iron levels of approximately 80 µmol iron per gram of liver, wet weight (»15 mg iron per gram of liver, dry weight), are at an increased risk of hepatic disease and endocrine organ damage. Patients with higher body iron burdens are at particular risk of cardiac disease and early death.
Disordered iron metabolism is less common in the adult forms of a thalassemia. The reason is not clear, but the milder degree of anemia, fewer transfusions, and the less marked erythroid expansion of the marrow are the likely explanations.
There appears to be an increased susceptibility to bacterial infection in all forms of severe thalassemia.7,10,177,183 The reason is not known. It has been suggested that the relatively high serum iron levels may favor bacterial growth. Another possible mechanism is blockade of the monocyte-macrophage system due to the increased rate of destruction of red cells. No consistent defects in white cell or immune function have been demonstrated, and it remains to be demonstrated unequivocally that high serum iron levels are an important factor. The one exception is infection with Yersinia enterocolitica, a normally nonvirulent pathogen that is able to produce its own siderophore and hence that thrives in iron excess.
COAGULATION DEFECTS
The increasing knowledge about the potential hypercoagulable state in some forms of thalassemia has been reviewed in detail.166,167 and 168 There is some evidence that patients, particularly after splenectomy and with high platelet counts, may develop progressive pulmonary arterial disease due to platelet aggregation in the pulmonary circulation. Furthermore, using thalassemic red cells as a source of phospholipids, enhanced thrombin generation has been demonstrated in a thrombinase assay. The procoagulant effect of thalassemia cells appears to be due to an increased expression of anionic phospholipids on the red cell surface. Normally, neutral or negatively charged amino acids are confined to the inner leaflet of the red cell membrane, an effect that is mediated by the action of aminophospholipid translocase, an enzyme sometimes known as flipase, which, in effect, flips aminophospholipids that are diffused to the outer leaflet back to the inner leaflet (see Chap. 28). It is believed that in thalassemic red cells these aminophospholipids are moved to the outer leaflet and thus provide a surface on which coagulation can be activated. Other nonspecific changes in the coagulation pathway and its antagonists have also been observed in patients with different forms of thalassemia.
CLINICAL HETEROGENEITY
The pathophysiologic mechanisms described in the previous sections provide the basis for the remarkable diversity of the clinical findings in the thalassemia syndromes. It is apparent that all the manifestations of b thalassemia can be related to excess a-chain production. It follows that any mechanism that reduces the excess of a chains should reduce the clinical severity of the disease. An elegant “experiment of nature” has shown that this is so and, incidentally, has confirmed that globin-chain imbalance is the major factor in determining the severity of the thalassemias.
Coinheritance of a thalassemia can reduce the severity of the more severe forms of b thalassemia.184,185 and 186 This effect is much more marked in individuals who are homozygotes or compound heterozygotes for different forms of b+ thalassemia; bo-thalassemia homozygotes who have inherited a thalassemia seem to be protected little, if at all.
Severe b thalassemia can be modified by the coinheritance of genetic determinants for enhanced production of g chains. The interaction of the heterocellular HPFH in the amelioration of homozygous b thalassemia has already been mentioned. Other determinants may also be involved. For example, it is apparent that the inheritance of a particular RFLP haplotype in the region 5′ to the b-globin gene may be an important factor.187,188 This particular b-globin gene haplotype is associated with a single base change, C ® T, at position –158 relative to the Gg-globin gene, an alteration that creates a cleavage site for the restriction enzyme Xmn I.122 There is an excess of individuals with the –158 polymorphism with the phenotype of thalassemia intermedia compared with thalassemia major in different populations.188,189 It is still not absolutely clear, however, whether this polymorphism is the only factor in increasing hemoglobin F production in these cases. However, the association certainly points to an effect cis to the affected b gene as the basis for the elevated fetal hemoglobin levels in these forms of thalassemia intermedia.
Finally, some of the mutations that cause b thalassemia are associated with a mild phenotype because they result in only a modest reduction of b-chain production. For example, those at –29 and –88 have been found in association with mild b+ thalassemia in Africans. Similarly, in Mediterranean populations particularly mild phenotypes are found commonly with a base substitution at position 6 in IVS-1 and at position –87 in the 5′-flanking region of the b-globin gene. The homozygous state for the IVS-1 position 6 mutation usually produces an extremely mild form of b thalassemia. When these “mild” mutations are coinherited with more severe b-thalassemia determinants, the compound heterozygous states are characterized by a more severe form of thalassemia intermedia.
Other forms of thalassemia intermedia are associated with the homozygous state for db thalassemia, the various interactions of db thalassemia with b thalassemia, and heterozygous b thalassemia of the severe variety or in association with triplicated a-gene loci.7,10,190 These complex interactions are the subject of several extensive reviews.190,191 and 192
POPULATION GENETICS7,9,10,193
The b thalassemias are distributed widely in Mediterranean populations, the Middle East, parts of India and Pakistan, and throughout Southeast Asia (Fig. 46-13). The disease is common in the southern parts of the former USSR and in the People’s Republic of China. The b thalassemias are rare in Africa, except for some isolated pockets in West Africa, notably Liberia, and in parts of North Africa. However, b thalassemia occurs sporadically in all racial groups and has been observed in the homozygous state in persons of pure Anglo-Saxon heritage. Thus, a patient’s racial background does not preclude the diagnosis.

FIGURE 46-13 The world distribution of b thalassemia.

The db thalassemias have been observed sporadically in many racial groups, although no high-frequency populations have been defined. Similarly, the hemoglobin Lepore syndromes have been found in many populations, but, with the possible exceptions of central Italy, eastern Europe, and parts of Spain and Portugal, these disorders have not been found to occur with a high frequency in any particular region.
The a thalassemias occur widely throughout Africa, the Mediterranean countries, the Middle East, and Southeast Asia (Fig. 46-14). The ao thalassemias are found most commonly in Mediterranean and Oriental populations and are extremely rare in Africa and the Middle East. However, the deletion forms of a+ thalassemia occur with a high frequency throughout West Africa, the Mediterranean, the Middle East, and Southeast Asia. Up to 80 percent of the population of some parts of Papua New Guinea are carriers for the deletion form of a+ thalassemia. It is uncertain how common the nondeletion forms of a+ thalassemia are in any particular populations, but they have been reported quite frequently in some of the Mediterranean island populations and in the Middle East and Southeast Asia. Because the hemoglobin Bart’s hydrops syndrome and hemoglobin H disease require the action of an ao-thalassemia determinant, these disorders are found at a high frequency only in Southeast Asia and in parts of the Mediterranean region. The a-chain termination mutants, such as hemoglobin Constant Spring, seem to be particularly common in Southeast Asia, and in Thailand approximately 4 percent of the population are carriers.

FIGURE 46-14 The world distribution of a+ and ao thalassemia.

In 1949, J.B.S. Haldane suggested that thalassemia might have reached its high frequency in tropical regions because heterozygotes are protected against malaria.194 Although many population studies have been carried out in order to test this hypothesis, it is only in recent years, with the advent of recombinant DNA technology, that it has been possible to elucidate some of the extremely complex population genetics that underlie polymorphic systems such as the thalassemias.
It is now apparent that in each of the high-frequency areas for the b thalassemias there are a few common mutations, together with varying numbers of rare ones (see Fig. 46-13). Furthermore, in each of these regions the pattern of mutations is different, and, even when the same mutation occurs in different populations, it is usually found together with a different arrangement of RFLPs (haplotype) in the associated b-globin gene cluster.193,195,196 Similar observations have been made in the case of the a thalassemias (see Fig. 46-14).193 These studies suggest that the thalassemias arose independently in different populations and then achieved their high frequency by selection. Although there may have been some movement of the thalassemia genes by drift, there is little doubt that independent mutation and selection provides the overall basis for their world distribution. Early studies in Sardinia, which showed that b thalassemia is less common in the mountainous regions where malarial transmission is low, supported Haldane’s suggestion that b thalassemia might have reached its high frequency due to protection against malarial infections. For many years these data remained the only convincing evidence for such a protective effect.197 However, more recent studies making use of malaria endemicity data together with globin-gene mapping have shown a very clear altitude-related effect on the frequency of a thalassemia in Papua New Guinea. In addition, a sharp cline in the frequency of a thalassemia has been found in the region stretching south from Papua New Guinea through the island populations of Melanesia to New Caledonia; this is mirrored by a similar gradient in the distribution of malaria.198 The effect of drift and founder effect in these island populations has been largely excluded by showing that other DNA polymorphisms have a random distribution through the region, with no evidence of a cline similar to that which characterizes the distribution of a thalassemia and malaria.
More recently, it has been possible to provide firm evidence for the protection of individuals with mild forms of a+ thalassemia against Plasmodium falciparum malaria. In a case control study carried out in Papua New Guinea, it was found that the homozygous state for a+ thalassemia offered approximately 60 percent protection against being admitted to hospital with serious complications of malaria, notably coma or profound anemia.199 Furthermore, long-term follow-up studies of cohorts of babies carried out in the Vanuatan islands showed, surprisingly, that, in the first years of life, those who are homozygous for a+ thalassemia are more prone to malaria, both Plasmodium vivax and P. falciparum.200 This surprising finding is of particular interest from the mechanistic viewpoint. It suggests that a thalassemic babies may be more prone to infection at a time when they are less likely to die of malaria because they are protected by other mechanisms; this may induce an early immunization that results in later protection. This concept is supported by the observation that these babies tend to get the mild P. vivax infections earlier than those due to P. falciparum; there is increasing evidence that there may be cross-immunization between the two species.
A great deal of work has been carried out toward understanding the reason why thalassemic red cells appear to be protective against malarial parasites. A variety of studies have failed to demonstrate any effect of the thalassemia phenotype on the rates of parasite invasion and growth.201,202 However, parasitized a-thalassemic cells bind significantly more antibody from the serum of patients with acute P. falciparum malaria than do normal red cells.203 It is not yet clear whether this reflects more efficient exposure of malarial antigens by the thalassemic cells or whether these cells expose red cell neoantigens related to senescence more effectively than do normal cells when invaded by the parasite. This phenomenon has also been observed in parasitized b-thalassemic cells, and these observations raise a new avenue of investigation for the protective effect of thalassemia against P. falciparum malaria. In effect, they suggest, like the population studies outlined earlier, that it may be immune mediated rather than due to the particular properties of the small thalassemic red cells themselves. Indeed, several lines of evidence now suggest that parasitized thalassemic cells may be more prone to ingestion by macrophages.202
CLINICAL FEATURES
b AND db THALASSEMIAS
The most clinically severe form of b thalassemia is called thalassemia major. A milder clinical picture, characterized by a later onset and either no transfusion requirement or at least fewer transfusions than are required to treat the major form of the illnesses, is designated b thalassemia intermedia. b Thalassemia minor is the term used to describe the heterozygous carrier state for b thalassemia.
b THALASSEMIA MAJOR7,10,177
The homozygous or compound heterozygous state for b thalassemia, thalassemia major, produces the clinical picture first described by Cooley in 1925.1 Affected infants are well at birth. Anemia usually develops during the first few months of life and becomes progressively more severe. These infants fail to thrive and may have feeding problems, bouts of fever, and diarrhea and other gastrointestinal symptoms. The majority of infants who will develop transfusion-dependent homozygous b thalassemia present with these symptoms within the first year of life. A later onset suggests that the condition may develop into one of the intermediate forms of b thalassemia (see “b Thalassemia Intermedia”).
The course of the disease in childhood depends almost entirely on whether the child is maintained on an adequate transfusion program. The classic textbook picture of Cooley’s anemia describes the disease as it was seen before these children could be maintained with relatively normal hemoglobin levels by regular blood transfusions. If adequate transfusion is possible, children grow and develop normally and have no abnormal physical signs. Few of the complications of the disorder occur during childhood, and the disease presents a problem only when the effects of iron loading resulting from ineffective erythropoiesis and from repeated blood transfusions begin to become apparent at the end of the first decade. Those who are treated with an adequate iron chelation regimen develop normally, although some of them remain short in height.
An inadequately transfused child develops the typical features of Cooley’s anemia. Growth is stunted, and, with bossing of the skull and overgrowth of the maxillary region, the face gradually assumes a “mongoloid” appearance. These changes are associated with a characteristic radiologic appearance of the skull, long bones, and hands (Fig. 46-15). There is widening of the diploe, with a “hair on end” or “sun-ray” appearance and a lacy trabeculation of the long bones and phalanges, and there may be gross skeletal deformities. The liver and spleen are enlarged, and the pigmentation of the skin increases. Many features of a hypermetabolic state with fever, wasting, and hyperuricemia may develop.

FIGURE 46-15 Radiologic appearances of the hands in homozygous b thalassemia.

The clinical course is characterized by severe anemia with frequent complications. These children are particularly prone to infection, which is a common cause of death. Because of increased folate utilization by the hypertrophied marrow, folic acid deficiency occurs frequently. Spontaneous fractures occur commonly as a result of the expansion of the marrow cavities with thinning of the long bones and skull. Maxillary deformities often lead to dental problems from malocclusion. The formation of massive deposits of extramedullary hematopoietic tissue may cause neurologic complications. With the gross splenomegaly that may occur, secondary thrombocytopenia and leukopenia frequently develop, leading to a further tendency to infection and bleeding. There may be a bleeding tendency in the absence of thrombocytopenia. Epistaxis is particularly common. These hemostatic problems are associated with poor liver function in some cases. Chronic leg ulceration may occur, although this is more common in thalassemia intermedia.
Children who have grown and developed normally throughout the first 10 years of life as a result of regular blood transfusion begin to develop the symptoms of iron loading as they enter puberty, particularly if they have not received adequate iron chelation.177 The first indication of iron loading is usually the absence of the pubertal growth spurt and a failure of the menarche. Over the succeeding years, a variety of endocrine disturbances may develop, particularly diabetes mellitus, hypogonadotrophic hypogonadism, and growth hormone deficiency; hypothyroidism and adrenal insufficiency also occur but are less common.179 Toward the end of the second decade, cardiac complications arise, and death usually occurs in the second or third decade as a result of cardiac siderosis. This may cause an acute cardiac death with arrhythmia, or intractable cardiac failure. Both of these complications may be precipitated by intercurrent infection.
Even the adequately transfused child who has received chelation therapy may suffer a number of complications. Blood-borne infection, notably with hepatitis C204 or HIV,205 are extremely common in some populations, although their frequency is being reduced by the use of widespread blood-donor screening programs. Delayed puberty and growth retardation are also common and probably reflect hypogonadotrophic hypogonadism together with damage to the pituitary gland.204,206 Osteoporosis is also being recognized increasingly and may also be, at least in part, a reflection of hypogonadism.204
b THALASSEMIA INTERMEDIA
The clinical phenotype of patients designated to have thalassemia intermedia is more severe than the usual asymptomatic thalassemia trait but milder than transfusion-dependent thalassemia major.7,10,190,191 and 192 The syndrome encompasses disorders with a wide spectrum of disability. At the severe end, patients present with anemia later than is usual in the transfusion-dependent forms of homozygous b thalassemia and are just able to maintain a hemoglobin level of about 6 g/dl without transfusion. However, their growth and development are retarded, and they become seriously disabled, with marked skeletal deformities, arthritis, and bone pain; progressive splenomegaly; growth retardation; and chronic ulcerations above the ankles. At the other end of the spectrum, there are patients who remain completely asymptomatic until adult life and are transfusion-independent, with hemoglobin levels as high as 10 to 12 g/dl. All varieties of intermediate severity are observed, and some patients become disabled simply due to the effects of hypersplenism. Intensive studies of the molecular pathology of this condition have provided some guidelines about genotype-phenotype relationships that are useful for genetic counseling (Table 46-7).

TABLE 46-7 b THALASSEMIA INTERMEDIA

b THALASSEMIA MINOR
The heterozygous state for b thalassemia is not usually associated with any clinical disability, and the abnormality is discovered only on performing a blood examination. It is most commonly discovered during periods of stress, such as pregnancy or during severe infection, when a moderate degree of anemia may be found. Some patients with thalassemia minor have increased iron stores, but often this may be due to injudicious iron therapy started because of misdiagnosed microcytic anemia.
a THALASSEMIAS
THE HEMOGLOBIN BART’S HYDROPS FETALIS SYNDROME
This disorder is a frequent cause of stillbirth in Southeast Asia. Infants either are stillborn between 34 and 40 weeks’ gestation or are born alive but die within the first few hours.207,208 There is pallor, edema, and hepatosplenomegaly, and the clinical picture resembles hydrops fetalis due to Rh blood group incompatibility. At autopsy there is massive extramedullary hemopoiesis and enlargement of the placenta. A variety of congenital anomalies have been observed.
There have been a few reports of the rescue of infants with this syndrome by prenatal detection and exchange transfusion. These babies have grown and developed normally, although they are of course blood transfusion–dependent.208,209 and 210
This condition is associated with a high incidence of maternal toxemia of pregnancy and difficulties at the time of delivery because of the massive placenta.7,207,208 The reason for placental hypertrophy is unknown, although, because a similar phenomenon is observed in hydrops infants with Rh incompatibility, it may reflect severe intrauterine hypoxia.
HEMOGLOBIN H DISEASE
Hemoglobin H disease was described independently in the United States and in Greece in 1956.211,212 The clinical findings are variable; a few patients are almost as severely affected as patients with b thalassemia major, while most have a much milder course.7,10,172,213 There is lifelong anemia with variable splenomegaly; bone changes are unusual.
There have been a few attempts to correlate the genotype with the phenotype of hemoglobin H disease. In general, it appears that, as might be expected, patients with a nondeletion form of a thalassemia affecting the predominant a2 gene interacting with an ao thalassemia determinant (– –/aTa), – –/a Constant Springa, for example, have higher levels of hemoglobin H, a greater degree of anemia, and, anecdotally, a more severe clinical course than patients with the ––/–a genotype.214,215,216 and 217
MILDER FORMS OF a THALASSEMIA, INCLUDING THE TRAITS
Because there are two a-globin genes per haploid genome, there is a wide spectrum of different conditions with overlapping phenotypes resulting from their various interactions. The carrier states for the deletion and nondeletion forms of a thalassemia, –a/aa and aT/a/aa, are symptomless. Similarly, the homozygous states for the deletion forms of a+ thalassemia, –a/–a, and the heterozygous state for ao thalassemia, – –/aa, are symptomless, although they are associated with mild anemia and red cell changes. On the other hand, the homozygous states for the nondeletion forms of a thalassemia, aTa/aTa, are associated with an extremely diverse series of phenotypes. As mentioned earlier, in the section on Interactions of a-Thalassemia Haplotypes, they sometimes result in the clinical picture of hemoglobin H disease, while in others they may be associated with only a mild hypochromic anemia.7 The homozygous states for the chain termination mutants, notably hemoglobin Constant Spring, constitute a special case because they produce a particularly characteristic phenotype. In this case there is a moderate hemolytic anemia with splenomegaly and characteristic hematological findings.218,219 and 220
LABORATORY FEATURES
b THALASSEMIA MAJOR
Hemoglobin levels at presentation may be in the range of 2 to 3 g/dl or even lower. The red cells show marked anisopoikilocytosis, with hypochromia, target cell formation, and a variable degree of basophilic stippling (Fig. 46-16). The appearance of the blood film varies somewhat, depending on whether the spleen is intact. In nonsplenectomized patients, large poikilocytes are common, whereas after splenectomy, large, flat macrocytes and small, deformed microcytes are frequently seen. The reticulocyte count is moderately elevated, and there are nearly always nucleated red cells in the blood. These red cells may reach very high levels after splenectomy. The white cell and platelet counts are slightly elevated unless there is secondary hypersplenism. Staining of the blood with methyl violet, particularly in splenectomized subjects, reveals stippling or ragged inclusion bodies in the red cells.160 These inclusions can nearly always be found in the red cell precursors in the marrow (Fig. 46-17). The marrow usually shows erythroid hyperplasia with morphologic abnormalities of the erythroblasts such as striking basophilic stippling and increased iron deposition. Iron kinetic studies indicate that there is markedly ineffective erythropoiesis, and red cell survival is usually shortened. There are populations of cells with very short survival and also longer-lived populations of cells; the latter contain relatively more fetal hemoglobin.173,174 An increased level of fetal hemoglobin, ranging from less than 10 percent to over 90 percent, is characteristic of homozygous b thalassemia. In bo thalassemia, no hemoglobin A is produced. The acid elution test shows that the fetal hemoglobin is quite heterogeneously distributed among the red cells. Hemoglobin A2 levels in homozygous b thalassemia may be low, normal, or high. If expressed as a proportion of hemoglobin A, however, the hemoglobin A2 level is almost invariably elevated. Differential centrifugation studies indicate some heterogeneity of hemoglobin F and A2 distribution among thalassemic red cells, and their level in whole blood gives little indication of their total rates of synthesis.

FIGURE 46-16 The peripheral blood film in homozygous b thalassemia.

FIGURE 46-17 Red cell inclusions in peripheral blood of a homozygous b thalassemia patient (postsplenectomy).

In vitro hemoglobin synthesis studies using marrow or blood show a marked degree of globin-chain imbalance; there is always a marked excess of a over b and g-chain production.158,159 Other aspects of the laboratory findings in this condition, including red cell survival, iron absorption, ferrikinetics, erythrokinetics, and the consequences of iron loading, are discussed earlier (see “Pathophysiology”).
b THALASSEMIA MINOR
Hemoglobin values of patients with b thalassemia minor are usually in the range of 9 to 11 g/dl. The most consistent finding is small, poorly hemoglobinized red cells, resulting in MCH values of 20 to 22 pg and MCV values of 50 to 70 fl. The red cell indices are particularly useful in screening for heterozygous carriers of thalassemia in population surveys. The marrow in heterozygous b thalassemia shows slight erythroid hyperplasia with rare red cell inclusions. Megaloblastic transformation due to folic acid deficiency occurs occasionally, particularly during pregnancy. There is a mild degree of ineffective erythropoiesis, but red cell survival is normal or nearly so. The hemoglobin A2 level is increased to 3.5 to 7 percent. The level of fetal hemoglobin is elevated in about 50 percent of cases, usually to 1 to 3 percent and rarely to more than 5 percent.
a THALASSEMIAS
THE HEMOGLOBIN BART’S HYDROPS FETALIS SYNDROME
In infants with the hydrops fetalis syndrome, the blood film shows severe thalassemic changes with many nucleated red cells. The hemoglobin consists mainly of hemoglobin Bart’s, with approximately 10 to 20 percent hemoglobin Portland. There is usually no hemoglobin A or F, although the rare cases that seem to result from the interaction of ao thalassemia with a severe nondeletion form of a+ thalassemia show small amounts of hemoglobin A.
HEMOGLOBIN H DISEASE
The blood film shows hypochromia and anisopoikilocytosis. The reticulocyte count is usually in the 5 percent range. Incubation of the red cells with brilliant cresyl blue results in ragged inclusion bodies in practically all the cells. These form because of precipitation of hemoglobin H in vitro as a result of redox action of the dye. After splenectomy, large, single Heinz bodies are observed in some cells (Fig. 46-18). These are formed by the in vitro precipitation of the unstable hemoglobin H molecule and are seen only after splenectomy. Hemoglobin H constitutes between 5 and 40 percent of the total hemoglobin; there may also be traces of hemoglobin Bart’s, and the level of hemoglobin A2 is usually slightly subnormal.

FIGURE 46-18 Hb H disease: (A) blood film; (B) preformed inclusions postsplenectomy; (C) inclusions generated by brilliant cresyl blue.

a0-THALASSEMIA AND a+-THALASSEMIA TRAITS
The ao-thalassemia trait is characterized by the presence of 5 to 15 percent hemoglobin Bart’s at birth.7,125 This hemoglobin disappears during maturation and is not replaced by a similar amount of hemoglobin H. An occasional cell with hemoglobin H inclusion bodies may appear after incubation with brilliant cresyl blue, and this phenomenon is often used as a diagnostic test for the a-thalassemia trait. However, it is difficult to standardize and requires much experience to be useful. In adult life, the red cells of heterozygotes have morphologic changes of heterozygous thalassemia with low MCH and MCV values. The electrophoretic pattern is normal, and globin-synthesis studies show a deficit of a-chain production, with an a/b-chain production ratio of approximately 0.7.7,125
The a+-thalassemia trait is characterized by minimal hematologic changes, 1 to 2 percent of hemoglobin Bart’s at birth in some but not all cases, and a slightly reduced a/b-chain production ratio of approximately 0.8. This ratio can be distinguished from normal only by studying relatively large numbers of samples and comparing the mean a/b ratio with that of normal control subjects. This approach is not reliable for diagnosing individual cases of the a+-thalassemia trait, and, unfortunately, there is no really reliable way of making the diagnosis in adults except by DNA analysis.
Studies using DNA analysis indicate that there is a marked overlap between the different a-thalassemia carrier states with regard to the hematologic and globin-synthesis findings (reviewed in Ref. 7 and 125). In addition, they show that many a+-thalassemia carriers do not have elevated levels of hemoglobin Bart’s at birth. These studies confirm that, short of gene-mapping analysis, there is no way to identify specific a-thalassemia carrier states with certainty.
HOMOZYGOUS STATE FOR NONDELETION TYPES OF a THALASSEMIA
The homozygous state for nondeletion forms of a thalassemia involving the dominant (a2) globin gene causes a more severe deficit of a chains than do the deletion forms of a+ thalassemia. In some cases it produces hemoglobin H disease. The homozygous state for hemoglobin Constant Spring or other chain-termination mutations is associated with a moderately severe hemolytic anemia in which, for reasons that are not explained, there is no hemoglobin H but there are small amounts of hemoglobin Bart’s that persist into adult life. The homozygous states for the other nondeletion forms of a+ thalassemia are associated with hemoglobin H disease.
In the homozygous state for hemoglobin Constant Spring, the blood picture shows mild thalassemic changes with normal-sized red cells.218,219 and 220 The hemoglobin consists of about 5 to 6 percent hemoglobin Constant Spring, normal levels of hemoglobin A2, and trace amounts of hemoglobin Bart’s. The remainder is hemoglobin A.
The heterozygous state for hemoglobin Constant Spring shows no hematologic abnormality. The hemoglobin pattern is normal except for the presence of approximately 0.5 percent hemoglobin Constant Spring. The latter can be observed on alkaline starch-gel electrophoresis as a faint band migrating between hemoglobin A2 and the origin. It is best seen on heavily loaded starch gels and is easily missed if other electrophoretic techniques are used (Fig. 46-19). In the newborn period there is usually 1 to 3 percent hemoglobin Bart’s in the cord blood.

FIGURE 46-19 Hb Constant Spring. Starch gel electrophoresis of the following (left to right): 1 and 2, normal adult; 3 and 4, compound heterozygotes for Hb Constant Spring and ao thalassemia with Hb H disease; 5, normal adult; 6, compound heterozygote for ao thalassemia and Hb Constant Spring.

HOMOZYGOUS STATE FOR DELETION FORMS OF a+ THALASSEMIA
The homozygous state for deletion forms of a+ thalassemia is characterized by a thalassemic blood picture with 5 to 10 percent hemoglobin Bart’s at birth and hematologic findings similar to those in ao-thalassemia heterozygotes in adult life.125 In general, the –a4.2 deletion is associated with a more severe phenotype than is the – –3.7 deletion.221
DIFFERENTIAL DIAGNOSIS
COMMON FORMS OF THALASSEMIA
The clinical and hematologic findings in cases of homozygous b thalassemia and hemoglobin H disease are so characteristic that little difficulty in diagnosis is usually encountered. A simple flowchart for laboratory investigations of a suspected case is shown in Fig. 46-20.

FIGURE 46-20 A flowchart showing an approach to the diagnosis of the thalassemia syndromes.

In early childhood, difficulty may occasionally be encountered in distinguishing the thalassemias from the congenital sideroblastic anemias, but the marrow appearances in the latter are quite characteristic. Because of the high levels of hemoglobin F encountered in juvenile chronic myelogenous leukemia, this disorder may superficially resemble b thalassemia. However, the finding of primitive cells in the marrow and, on hemoglobin electrophoresis, the absence of elevated hemoglobin A2 levels and the decrease in carbonic anhydrase in juvenile chronic myelogenous leukemia readily differentiate this disorder from b thalassemia.
LESS COMMON FORMS OF THALASSEMIA
(db)0 THALASSEMIA
The homozygous state for db thalassemia is clinically milder than Cooley’s anemia and is one form of thalassemia intermedia.222,223 and 224 Only hemoglobin F is present, and hemoglobins A and A2 are not produced. Heterozygous db thalassemia is hematologically similar to b thalassemia minor.7 The fetal hemoglobin level is higher, being in the 5 to 20 percent range, and the hemoglobin A2 value is normal or slightly reduced. As in b thalassemia, the fetal hemoglobin is heterogeneously distributed among the red cells, thus distinguishing this disorder from hereditary persistence of fetal hemoglobin (Fig. 46-21).

FIGURE 46-21 Acid elution preparations of blood films from the following (top to bottom): (A) d b thalassemia; (B) hereditary persistence of fetal hemoglobin; (C) an artificial mixture of fetal and adult red cells.

Heterozygosity for both b thalassemia and db thalassemia results is a condition clinically similar to but milder than Cooley’s anemia; the hemoglobin consists largely of hemoglobin F, with a small amount of hemoglobin A2. This occurs because the associated b-thalassemia gene has usually been the b0 variety. db Thalassemia has also been observed in individuals heterozygous for hemoglobin S or C.7
(db)+ THALASSEMIA AND THE HEMOGLOBIN LEPORE DISORDERS
The hemoglobin Lepore disorders have been described in the homozygous state and in the heterozygous state either alone or in association with b or db thalassemia, hemoglobin S, or hemoglobin C.7,10,225 In the homozygous state, approximately 20 percent of the hemoglobin is of the Lepore type and 80 percent is fetal hemoglobin; hemoglobins A and A2 are absent. The clinical picture is variable, with some cases being identical to transfusion-dependent homozygous b thalassemia and others being associated with the clinical picture of thalassemia intermedia. In the heterozygous state, the findings are similar to those of b thalassemia minor, the hemoglobin consisting of about 8 percent hemoglobin Lepore, with a reduced level of hemoglobin A2 and a slight but consistent increase in the level of fetal hemoglobin. The Lepore hemoglobins have been found sporadically in most racial groups. In the majority of cases, chemical analysis has shown that they are identical to hemoglobin Lepore Washington-Boston; hemoglobin Lepore Hollandia and Lepore Baltimore have been observed in only a few patients.7,225
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
What is known about the molelcular pathology of HPFH was described earlier in this chapter, and the currently accepted classification and nomenclature of this complex group of conditions are summarized in Table 46-4. The different forms of HPFH are of very little clinical importance except insofar as they may interact with thalassemia or the structural hemoglobin variants.
(db)o HPFH Homozygotes for (db)o HPFH have 100 percent hemoglobin F, and their blood shows mild thalassemic changes with reduced MCH and MCV values very similar to those observed in heterozygous b thalassemia. Hemoglobin synthesis studies show that such persons have imbalanced globin-chain production, with ratios similar to those observed in b-thalassemia heterozygotes.226 Heterozygotes have approximately 20 to 30 percent hemoglobin F with slightly reduced hemoglobin A2 values and completely normal blood pictures. Thus, it appears that this condition is an extremely well compensated form of db thalassemia in which the output g chains almost but not entirely compensates for the complete absence b and d chains. The different molecular forms of this condition show no difference in phenotype except in the proportion of Gg chains. The African forms of (db)o HPFH have been found in association with hemoglobins S and C or with b thalassemia. These compound heterozygous states are associated with little clinical disability.7,9,10
Gg(Agb)+ HPFH and Hemoglobin Kenya The deletion form of HPFH associated with the production of the Agb gene product found in hemoglobin Kenya has been observed only in heterozygotes.108,109 Such individuals have a completely normal blood picture and, as well as having 5 to 20 percent hemoglobin Kenya, have elevated levels of hemoglobin F in the 5 to 10 percent range; only Gg chains are present.
Nondeletion Types of HPFH Many nondeletion forms of HPFH associated with point mutations upstream from the g-globin genes have been described (see Table 46-4). Ggb+ HPFH has been found in the heterozygous and compound heterozygous states with b-globin chain variants in African populations. There are no associated clinical or hematologic findings. Compound heterozygotes for Ggb+ HPFH and hemoglobins S or C produce 45 percent of the abnormal hemoglobin, 30 percent hemoglobin A, and 20 percent hemoglobin F containing only Gg chains.227,228
The most common form of nondeletion HPFH, Agb+ HPFH, is found in Greeks.229,230 and 231 In the homozygous state, there are no clinical or hematologic abnormalities; the hemoglobin findings are characterized by approximately 25 percent fetal hemoglobin and reduced levels of hemoglobin A2, in the 0.8 percent range.232 Heterozygotes, who are also hematologically normal, have 10 to 15 percent hemoglobin F, almost all of the Ag variety. Compound heterozygotes with b thalassemia have high levels of hemoglobin F and a clinical picture that is only slightly more severe than the b-thalassemia trait.
In the British form of Agb+ HPFH,233 heterozygotes have approximately 5 to 12 percent hemoglobin F, while homozygotes have approximately 20 percent. There are no associated hematologic abnormalities, although, surprisingly, in this form of nondeletion HPFH the hemoglobin F seems to be rather unevenly distributed among the red cells.
There is a heterogeneous group of conditions associated with the persistent production of small amounts of hemoglobin F in adult life. They are categorized under the general heading of heterocellular HPFH. Their clinical importance is that, when they are coinherited with different forms of b thalassemia, they may lead to a greater output of hemoglobin F and hence to a milder phenotype. This type of interaction should be suspected when one or other parent of a patient with b thalassemia intermedia has an unusually high level of hemoglobin F for the b-thalassemia trait. Similarly, it is sometimes possible to find unaffected lateral relatives or other family members with slightly elevated levels of hemoglobin F. However, until the gene loci involved in these conditions are determined, it is not possible to identify them with certainty.
b THALASSEMIA ASSOCIATED WITH b-CHAIN STRUCTURAL HEMOGLOBIN VARIANTS
The most clinically important associations of b thalassemia with b structural hemoglobin variants are sickle cell thalassemia, hemoglobin C thalassemia, and hemoglobin E thalassemia. In addition, there are many reported interactions of b thalassemia with rare structural variants.7,9,10
Sickle cell thalassemia224,225,226,227,228,229,230,231,232,233,234,235 and 236 occurs in parts of Africa and in the Mediterranean population, particularly in Greece and Italy. It has also been observed in the Middle East and in parts of India. The clinical consequences of carrying one gene for hemoglobin S and one gene for b thalassemia depend entirely on the type of b-thalassemia mutation. The sickle cell–bo thalassemia interaction is characterized by a clinical disorder that is very similar to sickle cell anemia. Similarly, the interaction of the sickle cell gene with the more severe forms of b+ thalassemia that are associated with a marked reduction in b-globin synthesis have a similar clinical phenotype. On the other hand, the interaction of the sickle cell gene with very mild forms of b+ thalassemia may be quite innocuous.7,236 The latter disorder is characterized by a mild anemia associated with splenomegaly and a hemoglobin concentration of approximately 30 percent hemoglobin S and 25 percent hemoglobin A with an elevated level of hemoglobin A2. In all these interactions, one parent shows the sickle cell trait, and the other the b thalassemia trait.
Hemoglobin C thalassemia is a mild hemolytic disorder associated with splenomegaly.7,9,10 Again, the hemoglobin pattern varies depending on whether the thalassemia gene is the b+ or bo type. This relatively innocuous condition has been recorded mainly in North Africa but is also found in West African populations. It is characterized by a mild hemolytic anemia and splenomegaly with a blood picture that shows the numerous target cells characteristic of all the hemoglobin C disorders.
Hemoglobin E thalassemia is one of the most important hemoglobinopathies in the world population.7,9,10,237,238,239 and 240 As was mentioned earlier, hemoglobin E is synthesized at a reduced rate and hence produces the clinical phenotype of a mild form of b thalassemia. Hence, when it is inherited together with b thalassemia—and most often this is a bo or severe b+thalassemia mutation in Southeast Asia and India—there is a marked deficit of b-chain production, and hence the clinical picture of severe b thalassemia. In fact, hemoglobin E thalassemia shows a remarkable variability in clinical expression,241 ranging from a mild form of thalassemia intermedia to a transfusion-dependent condition clinically indistinguishable from homozygous b thalassemia. The reasons for this variability of expression are not understood, although some of the factors involved may be identical to those that modify other forms of b thalassemia.
In the more severe cases of hemoglobin E thalassemia, there is severe anemia with growth retardation, leg ulcers, bone deformity, a marked tendency to infection, iron loading, and variable splenomegaly and hypersplenism. Large tumor masses composed of extramedullary erythropoietic tissue may cause a variety of compression syndromes, including a clinical picture that can closely mimic a cerebral tumor. Another curious picture that seems to be restricted to splenectomized patients is an obliterative occlusion of the pulmonary vasculature that is thought to be the result of an extremely high platelet count.242
The blood picture shows a typical thalassemic pattern, and the hemoglobin consists of E, F, and A2; there is usually no hemoglobin A because the bo thalassemias are particularly common in the parts of the world where hemoglobin E is found.
b THALASSEMIA WITH NORMAL HEMOGLOBIN A2 LEVEL
There are forms of b thalassemia in which heterozygotes have normal hemoglobin A2 levels. Their main clinical importance is that they can be confused with the more severe forms of a thalassemia in the heterozygous state and therefore may cause difficulties for genetic counseling and prenatal diagnosis. Based on hematologic studies, there are two main classes of “normal hemoglobin A2 b thalassemia,” called types 1 and 2.243 Type 1 is the “silent” form of b thalassemia, while type 2 is heterogeneous, many cases representing the compound heterozygous state for b thalassemia and d thalassemia.
Normal A2 b thalassemia type 1 is characterized by no hematologic changes in heterozygotes, and it can be identified with certainty only by globin-chain synthesis studies, which show mild chain imbalance with a/bglobin-chain synthesis ratios of approximately 1.3. The condition is also called silent b thalassemia.244 Compound heterozygotes for this condition and b thalassemia have a mild form of b thalassemia intermedia.
Normal hemoglobin A2 b thalassemia type 2 in heterozygotes is indistinguishable from typical b thalassemia with elevated hemoglobin A2 levels.243 The homozygous state has not been described. The compound heterozygous state for this gene and for b thalassemia with raised hemoglobin A2 levels is characterized by a clinical picture of severe transfusion-dependent b thalassemia. Family data obtained in Italy and Sardinia suggest that this condition usually represents the compound heterozygous state for both b thalassemia and d thalassemia.245,246 Most of the d thalassemias have been observed trans to b thalassemia. However, the form of d thalassemia that results from the loss of an A in codon 59 occurs on the same chromosome as the hemoglobin Knossos mutation, which is associated with a mild form of b thalassemia.247 This explains the normal level of hemoglobin A2 associated with this condition, which is the most common form of normal hemoglobin A2 b thalassemia in the Mediterranean region.
Several other conditions mentioned earlier in this chapter are associated with a phenotype that is indistinguishable from normal A2 b thalassemia. These include the heterozygous states for the Corfu form of db thalassemia, and egdb thalassemia.
OTHER UNUSUAL FORMS OF b THALASSEMIA
The clinical features of the dominant b thalassemias, the molecular pathology resembles closely that of thalassemia intermedia. There is moderate anemia and splenomegaly, together with a blood picture showing thalassemic red cell changes. The marrow shows erythroid hyperplasia with well-marked inclusion bodies in the red cell precursors; the latter may be seen in the peripheral blood after splenectomy. Hemoglobin analysis shows hemoglobins A and A2, and the level of hemoglobin F is not usually elevated much above that seen in b-thalassemia trait. The hemoglobin A2 levels are always raised.
Other unusual varieties of b thalassemia include those that are categorized by having unusually high hemoglobin F or A2 levels. It is now apparent that most of these conditions result from deletions that involve the b-globin gene and its promoter region. For example, the so-called Dutch248 form of b thalassemia is associated with unusually high levels of hemoglobin F in heterozygotes and high levels of hemoglobin A2; several other conditions of this type, due to different-sized deletions, have been reported.
d THALASSEMIA
d Thalassemia causes a complete absence of hemoglobin A2 in homozygotes and a reduction in the level of hemoglobin A2 in heterozygotes.249 Apart from its effect of reducing hemoglobin A2 levels in b-thalassemia heterozygotes, it is of no clinical significance.
egdb THALASSEMIA
This heterogeneous condition has been observed only in the heterozygous state in a few families.7,36,101,102 and 103 It is characterized by neonatal hemolysis and, in adult life, by the hematologic picture of heterozygous b thalassemia with normal hemoglobin A2 levels.
a THALASSEMIA IN ASSOCIATION WITH a- AND b-CHAIN HEMOGLOBIN VARIANTS
Several a-globin structural variants are due to single amino acid substitutions at a-chain loci on chromosomes that carry only a single a-chain gene. Individuals who inherit variants of this type together with an ao-thalassemia determinant have a form of hemoglobin H disease in which the hemoglobin consists of the a-chain variant hemoglobin and hemoglobin H. Well-documented examples include hemoglobin QH disease (– –/–aQ),250,251 hemoglobin G Philadelphia H disease (– –/–aG),252,253 and hemoglobin Hasharon H disease (– –/–aHash).254 There are many examples of the coexistence of the homozygous or heterozygous states for b-chain hemoglobin variants and different a-thalassemia determinants.7,9,10 Particularly well-characterized disorders include the various interactions of ao and a+ thalassemia with hemoglobin E7,237 and hemoglobin S.255,256 Carriers for these hemoglobin variants who also have the ao- or a+-thalassemia traits have thalassemic red cell indices and unusually low levels of the abnormal hemoglobin. Individuals with sickle cell anemia who have a thalassemia show thalassemic red cell changes, more persistent splenomegaly, and lower hemoglobin F values than do those without the thalassemia genes.255,256
THERAPY, COURSE, AND PROGNOSIS
The only forms of treatment available for thalassemic children are regular blood transfusions, iron chelation therapy in an attempt to prevent iron overload, the judicious use of splenectomy in cases complicated by hypersplenism, and a good standard of general pediatric care. Marrow transplantation also has an important role in selected cases (see Chap. 19).
TRANSFUSION
If children with b thalassemia are maintained at a hemoglobin level of 9.5 to 14 g/dl, they grow and develop normally and develop none of the distressing skeletal complications of thalassemia.7,257 More recent experience suggests that it may be possible to maintain a lower hemoglobin level than this without any deleterious effects on development and with the added advantage of reducing the level of iron loading. This regimen maintains a mean pretransfusion level that does not exceed 9.5 g/dl.258 The transfusion program should not be started too early, and only when it is quite clear that the hemoglobin level is too low to be compatible with normal development. If transfusion is started too soon, thalassemia intermedia may be missed, and the child may be transfused unnecessarily. Usually blood transfusions are given every four weeks on an outpatient basis. To avoid transfusion reactions, it is important to use washed, filtered, or frozen red cells so that the majority of the white cells and plasma-protein components are removed (see Chap. 142). More ambitious programs using separated, young erythrocyte populations (“neocytes”) for transfusion, together with the removal of the patient’s older cells, have been described,229 but their use is restricted to only a few centers because of the difficulty and expense of these procedures.257
IRON CHELATION
Since every child maintained on a high-transfusion regimen will ultimately develop iron overload and die due to siderosis of the myocardium, it is vital, when possible, to start such children on a program of iron chelation some time within the first 2 to 3 years of life.182 Despite extensive searches for an oral chelating agent, deferoxamine (desferrioxamine) is currently the only drug of proven value for the treatment of thalassemia. It is best administered by an 8- to 12-h overnight pump-driven infusion in the subcutaneous tissues of the anterior abdominal wall.260,261 Chelation therapy should commence by the time the serum ferritin level has reached approximately 1000 µg/dl. In practice this is usually after the twelfth to fifteenth transfusion. It is important not to overchelate infants when the iron burden is still low in order to avoid toxicity. The initial dose is usually 20 mg/kg on 5 nights a week, with 100 mg of oral vitamin C (200 mg in older children and adults) on the days of the infusion, since this increases the level of iron excretion.262 Higher levels of ascorbate should be avoided because of the potential for toxicity.263 In patients who are heavily iron loaded, particularly with cardiac or endocrine complications, the body iron stores may be effectively lowered by the use of a continuous intravenous infusion of desferrioxamine at a dose of up to 50 mg/kg body weight. This usually entails the insertion of an intravenous delivery system.
It has been usual to monitor the degree of iron loading by the use of serial serum ferritin estimations. However, recent studies indicate that the relationship between hepatic iron concentration and serum ferritin is not reliable, and it is recommended that all patients on regular transfusion are monitored with hepatic iron studies (see Iron Metabolism).257 If this is not possible, it is important to try to maintain the serum ferritin levels below 1500 µg/l.
There is extensive experience of the use of desferrioxamine and its toxic effects.257 Apart from local erythema and painful subcutaneous nodules at the site of infusions and occasional genuine allergic reactions, there are no serious complications, and these reactions can be controlled, at least in part, by inclusion of 5 to 10 mg hydrocortisone in the infusion. Probably of greatest concern is neurosensory toxicity, which has been documented in up to 30 percent of cases. This causes high-frequency hearing loss that may sometimes become symptomatic.264,265 In a few cases, this has not responded to discontinuation of the drug, and there has been permanent hearing loss. Ocular toxicity has also been reported.264 This involves visual failure, with night and color blindness together with field loss. Reversal after discontinuation of the drug has been reported. Desferrioxamine may also cause bone changes and growth retardation, sometimes associated with bone pain. Body measurements characteristically show a reduced crown-pubis/pubis-heel ratio. These changes may be associated with radiological abnormalities of the vertebral column. The occurrence of these complications can be avoided by extreme care in monitoring patients receiving long-term desferrioxamine therapy. It appears that young children or individuals from whom most of the iron has been removed by chelation are at particularly high risk. It is recommended that a formal audiometry and ophthalmologic examination is carried out at 6-month intervals.
The only oral iron chelating agent that has received extensive study is 1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone, L1). The current status of this drug has been reviewed recently.257 While early studies were promising, it is now apparent that, at the dose currently used, some patients do not maintain iron balance. About 5 percent of patients develop severe neutropenia, and there have been deaths from agranulocytosis. There is also concern about the possibility that this agent may potentiate liver fibrosis. The future role of this agent is not clear, but it certainly should be used only with very close monitoring and facilities for liver biopsy.
There is increasing evidence that children maintained at a high hemoglobin level do not develop hypersplenism. However, in patients who have been kept at a lower hemoglobin level, enlargement of the spleen with increased transfusion requirements occurs commonly. Splenectomy should be carried out if there is a dramatic increase in the transfusion requirements or pain develops because of the size of the spleen. Because of the risk of overwhelming pneumococcal infections,266,267 this should not be done in the first 5 years of life. Before it is carried out, these children should receive a pneumococcal vaccine, and then they should be placed on prophylactic oral penicillin after the operation. It is also recommended that they receive Haemophilus influenzae type B and meningococcal vaccines.
Children with severe thalassemia are still prone to other infections. Presentation with abdominal pain, diarrhea, and vomiting should always suggest the possibility of an infection with a member of the Yersinia class of bacteria. Empirical treatment should start immediately with either an aminoglycoside or a cotrimoxazole. Transfusion-transmitted virus infection is also very common in some populations. All chronically transfused patients should be tested for hepatitis C, hepatitis B, and HIV annually, and patients with serological evidence of chronic active hepatitis should be considered for treatment with interferon alpha and/or ribavirin. As mentioned earlier, there is increasing recognition of subtle endocrine deficiencies, particularly associated with growth retardation and hypogonadism. These patients require expert endocrinological assessment and, when appropriate, replacement therapy.
MARROW TRANSPLANTATION
By 1997, over 1000 marrow transplants had been performed at three centers in Italy.268,269,270 and 271 Based on early experiences, it became clear that the prognosis depended very much on the adequacy of iron chelation up to the time of transplantation. Based on this history, patients were divided into three classes: class I have a history of adequate iron chelation and neither liver fibrosis nor hepatomegaly; class II are characterized by having one or two of these characteristics; and class III have all three. Among children in class I who had undergone transplantation early in the course of the disease, disease-free survival was assessed at 90 to 93 percent, with a risk of mortality related to the procedure of 4 percent.268 For class II patients, which form the intermediate risk group, the survival and disease-free survival rates were 86 percent and 82 percent, respectively. For what is considered to be the high-risk group, that is, class III, the survival and disease-free survival rates are 62 and 51 percent, respectively. Apart from the immediate complications of severe infection in the posttransplant period, most of the problems relate to the development of acute or chronic graft-versus-host disease. It appears that the overall frequency of mild to severe grades ranges from 30 to 27 percent.272 The modification of preparative drug regimens has reduced the frequency of drug toxicity. The occurrence of mixed chimerism may be a risk factor for graft-versus-host disease. So far, the longest follow-up of patients after transplantation is between 15 and 20 years; no case of hematologic malignancy has been observed. A recent study suggests that it may be more effective to remove excess body iron accumulated before transplantation by venesection rather than chelation therapy.273
Clearly, in experienced centers, marrow transplantation now offers a genuine option for the management of different forms of thalassemia.
Apart from the measures just outlined, the management of thalassemia requires a high standard of general pediatric care. Infection should be treated early. If the diet is inadequate in folate, supplements should be given; this is probably unnecessary in children maintained on a high-transfusion regimen. Particular attention should be paid to the ear, nose, and throat because of the problem of chronic sinus infection and middle-ear diseases resulting from bone deformity of the skull. Similarly, regular dental surveillance is essential, since poorly transfused thalassemic children have a variety of deformities of the maxilla and poorly developed teeth. In the later stages of the illness, when iron loading becomes the major feature, endocrine replacement therapy may be necessary, together with symptomatic treatment for cardiac failure. It may be possible to improve cardiac function with intensive desferrioxamine therapy.
THERAPIES OF SPECIAL TYPES OF THALASSEMIA
Hemoglobin H disease usually requires no specific therapy, although splenectomy may be of value in cases associated with severe anemia and splenomegaly.7,9,10 This may be followed by a higher incidence of thromboembolic disease than occurs in splenectomized children with b thalassemia,7 and therefore the spleen should be removed only in cases of extreme anemia and splenomegaly. Oxidant drugs should be avoided in patients with hemoglobin H disease. The management of symptomatic sickle cell thalassemia follows the lines described for sickle cell anemia (see Chap. 48).
Thalassemia intermedia presents a particularly complex therapeutic problem. It is difficult to be certain whether a child with a steady-state hemoglobin level of 6 to 7 g/dl should be transfused. Probably the best compromise is to watch such children very closely during the first years of life, and, if they are growing and developing normally and there are no signs of bone changes, they should be maintained without transfusion. If, however, their early growth pattern is retarded or their activity is limited due to their anemia, they should be placed on a regular transfusion regimen. It is especially important to determine whether hypersplenism is playing a role in their anemia as they get older and to carry out splenectomy if this is the case. Since many of these patients have significant iron loading from the gastrointestinal tract, regular estimations of serum iron and ferritin should be carried out, and chelation therapy should be instituted when appropriate.257
EXPERIMENTAL APPROACHES TO TREATMENT
Two main experimental approaches are being pursued in the search for more effective therapy of the thalassemias: the reactivation or augmentation of fetal hemoglobin production and somatic gene therapy.
The main rationale for attempting to increase hemoglobin F production is based on the observation that patients recovering from cytotoxic drug therapy or during other periods of erythroid expansion may reactivate hemoglobin F synthesis. In addition, the observation that butyrate analogs might have a stimulating effect on hemoglobin F production has led to a number of studies of their potential for the management of thalassemia. A number of clinical trials have been summarized in recent reviews.274,275 and 276 Agents that have been used have included various cytotoxic drugs, erythropoietin, and several different butyrate analogs. Overall, while these agents, used either alone or in combination, have produced some small effects on fetal hemoglobin production, the results of these trials to date have been disappointing. There have been some notable exceptions, however, particularly several cases of homozygosity or compound heterozygosity for hemoglobin Lepore in which the use of either a combination of sodium phenylbutyrate and hydroxyurea or hydroxyurea alone has produced a spectacular rise in hemoglobin F production, which, in the case of two homozygotes for hemoglobin Lepore, removed the necessity for further transfusion.277 This raises the intriguing possibility that certain mutations, possibly deletions of the b-globin gene cluster, may be more susceptible to this type of approach. It may be that appropriate combination therapy will improve the results in other forms of thalassemia.
The other experimental approach involves somatic gene therapy. Currently this is mainly directed at gene transfer into potential hematopoietic stem cells using retroviral vectors,278 although other approaches are being taken, including attempts at the restoration of normal splicing in cases of splicing mutations279 or the use of trans-splicing ribozymes to correct b-globin gene transcripts.280,281 So far, none of these methods has been developed to the scale required for human gene transfer, and it looks as though it may be some time before this is achieved.
PROGNOSIS
There is now no doubt that the prognosis for patients with severe forms of b thalassemia who are adequately treated by transfusion and chelation has improved quite dramatically over recent years. Two large studies have investigated the influence of effective long-term use of desferrioxamine on the development of cardiac disease.282,283 In one trial, patients who had maintained sustained reduction of body iron, as estimated by a serum ferritin level of less than 2500 µg/l over 12 years of follow-up, had an estimated cardiac-disease–free survival of 91 percent, in contrast to patients in whom most determinations of serum ferritin level excluded this value, whose estimated cardiac-disease–free survival was less than 20 percent. In a second study, the relationship between survival and total body iron burden was measured directly using hepatic storage iron values. Patients who had maintained concentrations of hepatic iron equal to or exceeding 15 mg iron per gram of liver, dry weight, had a 32 percent probability of survival to the age of 25 years; no cardiac disease developed in patients who maintained hepatic iron levels below this threshold. These studies provide unequivocal evidence that adequate transfusion and chelation are now associated with longevity and a good quality of life. On the other hand, poor compliance or unavailability of chelating agents is still associated with a poor prospect of survival much beyond the second decade.
PREVENTION
In those parts of the world where the incidence is high, the economic burden placed on society by thalassemia is immense. For example, it was estimated that if all the thalassemic children who are born in Cyprus were treated by regular blood transfusions and iron chelating therapy, within 15 years the total medical budget of the island would be required to treat this single disease.284 Clearly, this approach is not always feasible, and hence there is considerable effort toward the development of programs for prevention of the different forms of thalassemia.
There are two ways in which this can be achieved. The first is by prospective genetic counseling, that is, screening total populations while still at school and warning carriers about the potential risks of marriage to another carrier. There are few data available about the value of programs of this type; a pilot study in Greece was unsuccessful,285 and the results of large-scale studies being carried out in parts of Italy286 are still awaited. Because it is felt that this approach is unlikely to be very successful in many populations, considerable effort has been directed toward developing prenatal diagnosis programs.
Prenatal diagnosis for the prevention of thalassemia entails screening mothers at the first prenatal visit, screening the father in cases in which the mother is a thalassemia carrier, and offering the couple the possibility of prenatal diagnosis and termination of pregnancy if they are both carriers of a gene for a severe form of thalassemia. Currently, these programs are devoted mainly to prenatal diagnosis of the severe transfusion-dependent forms of homozygous b+ or bo thalassemia. Considerable experience has also been gained in prenatal diagnosis of mothers at risk of having a fetus with the hemoglobin Bart’s hydrops syndrome because of the distress caused by a long and difficult pregnancy and the obstetric problems that result from the birth of a hydropic infant with a massive placenta.
The first efforts at prenatal detection of b thalassemia utilized fetal blood sampling and globin-chain synthesis analysis carried out at about the eighteenth week of pregnancy. Despite the technical difficulties involved, this method was applied successfully in many countries and has resulted in a reduction in the birth rate of infants with b thalassemia.287 It is associated with a low maternal morbidity rate, a fetal mortality rate of approximately 3 to 4 percent, and an error rate of 1 to 2 percent. Its main disadvantage is that it must be carried out relatively late in pregnancy. For this reason, efforts have turned to first-trimester prenatal diagnosis.
The application of the methods of DNA technology have made it possible to diagnose the important hemoglobin disorders in utero by fetal DNA analysis. Although this can be carried out on DNA derived from amniotic fluid, this approach has drawbacks because, again, it must be done relatively late in pregnancy, and often amniotic fluid cells have to be grown in culture to obtain enough DNA.288 However, it is possible to obtain DNA as early as the ninth week of pregnancy by chorionic villus sampling. Although the safety of this technique remains to be fully evaluated, and it is possible that limb reduction deformities may occur when the procedure is carried out very early in pregnancy (9 or 10 weeks), enough experience has been gained to suggest that it will become the major method for the prenatal diagnosis of the thalassemias.289,290,291 and 292
The identification of thalassemia in the fetus requires different approaches, depending on the nature of the molecular pathology involved.292 Major deletions, such as those that cause ao thalassemia and some of the bo thalassemias, can be identified directly on Southern blotting analysis of fetal DNA. About a third of the point mutations that produce b thalassemia alter restriction enzyme sites and therefore can also be identified by gene mapping. Where the mutation is known, oligonucleotide probes can be constructed to identify it directly. In families in which the mutation is not known, it is often possible to define the affected parental chromosomes by RFLP linkage analysis and then to determine whether the fetus has received both of the affected chromosomes from its parents. Experience of many hundred first-trimester prenatal diagnoses has suggested that it is possible to tell whether the fetus is affected in about 80 percent of cases.289,290,291 and 292 The main difficulties arise because many potentially affected fetuses will be compound heterozygotes for a common and a rare b-thalassemia mutation or because it may not be possible to define the thalassemia chromosome by RFLP analysis.
Now that the mutations have been determined in so many different forms of a and b thalassemia, it is possible to detect them directly as the first-line approach to prenatal diagnosis. Since most racial groups have only a few common b-thalassemia mutations, it is possible in many cases to determine the mutations in the parents and then to analyze fetal DNA for their presence. The development of PCR, combined with the use of oligonucleotide probes to detect individual mutations, offers a wide variety of new approaches for facilitating the speed and accuracy of carrier detection and prenatal diagnosis.293,294,295 and 296 For example, diagnoses can be made using hybridization of specific 32P end-labeled oligonucleotides to an amplified region of the b-globin gene dotted onto a nylon membrane. Because the b-globin gene sequence can be amplified more than 106-fold, hybridization time can be limited to 1 h, and the entire procedure can be carried out in 2 h. The ARMS (see Chap. 11) also allows the diagnosis to be made in about 2 h.297,298 Other modifications of PCR involve the use of non-radioactively labeled probes.299,300
The error rate using these different approaches varies, depending on a number of factors, particularly the experience of the laboratory. Low rates, less than 1 percent, have been reported from most laboratories using fetal DNA analysis. Potential sources of error include maternal contamination of fetal DNA, nonpaternity, and genetic recombination in cases where RFLP linkage analysis is used, along with other technical quirks.
The application of these approaches has caused a major reduction in the birth rate of infants with thalassemia in some populations, notably the Mediterranean islands. A number of methods are being explored to try to increase the options for prenatal detection of thalassemia. Harvesting of fetal cells from the maternal circulation is being explored, and a variety of ways are being investigated to isolate them by micromanipulation methods.301,302 Because of the trauma of termination of pregnancy experienced by many women, preimplantation approaches are also being explored. A few preimplantation diagnoses of b thalassemia by polar body analysis have already been carried out successfully.303
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Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

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