Physical Address
304 North Cardinal St.
Dorchester Center, MA 02124
Thalassemia Syndromes
The thalassemia syndromes are a heterogeneous group of inherited anemias which result from defects in the synthesis of one or more of the globin chain subunits of the hemoglobin (Hb) tetramer. The basic defect, decreased or absent production of a globin subunit, results in an imbalance between globins produced as a result of expression of genes from the alpha-globin gene cluster on chromosome 16, and those produced from expression of genes on the beta-globin gene cluster on chromosome 11. As a result of the imbalance, there is ineffective erythropoiesis (IE) and defective red cell production. The spectrum of disease severity is determined by the degree of imbalance in globin chain production and varies from mild, with few clinical manifestations, to severe anemia and transfusion dependence.
The disease was first described by Cooley and Lee in 1925, who coined the name Cooley’s anemia to describe a severe anemia with splenomegaly and bony changes. The term thalassemia is derived from a Greek term that roughly means “the sea” (Mediterranean) in the blood. It was first applied to the anemias frequently encountered in people from the Italian and Greek coasts and nearby islands, likely beta-thalassemia. The term thalassemia is now used to refer to a wide range of inherited defects in globin-chain biosynthesis. The inheritance of the disease, and its wide spectrum of severity was described progressively over the last century as our understanding of the disease and its pathophysiology has grown. Discovered to arise from abnormalities affecting expression of the autosomal globin genes, the homozygous condition was termed thalassemia major to describe the most severe form of the disease, the heterozygous condition was termed thalassemia minor or minima to describe a mostly asymptomatic carrier state, and the compound heterozygous states—somewhere in between—were called thalassemia intermedia.
The thalassemias are the commonest monogenic disorders in humans, and have a wide distribution across the globe. Approximately 1.5% of the world’s population carries one of the globin gene mutations, the majority of these individuals living in Asia, in areas of the world where thalassemia is particularly prevalent. It comprises a major public health problem. The main thalassemia syndromes are broadly alpha (α)-thalassemia and beta (β)-thalassemia, the clinical manifestations and morbidity of which vary based on the genotype and other modifying factors such as access to care and therapy-related complications, particularly iron overload.
This chapter reviews the major features of these syndromes. Readers wanting more detailed information than can be included here are referred to more comprehensive monographs elsewhere.
Definitions And Nomenclature
The most common forms of thalassemia arise from total absence of structurally normal globin chains or a partial reduction in their synthesis. In contrast to the “structural” hemoglobinopathies (e.g., sickle cell anemia), which are characterized by the production of normal amounts of mutant globin chains having abnormal physical or chemical properties, the thalassemias are quantitative disorders: the primary defects lie in the amount of globin produced. However, some forms of thalassemia are characterized by the production of structurally abnormal globin chains in reduced amounts, which are described in more detail in this chapter. These thalassemic hemoglobinopathies share features of thalassemia as well as those of structural hemoglobinopathies.
Individual syndromes are named according to the globin chain whose synthesis is adversely affected. Thus, α-globin chains are absent or reduced in patients with α-thalassemia, β-globin chains in patients with β-thalassemia, δ-globin and β-globin chains in patients with δβ-thalassemia, and so forth. In some contexts, it is also useful to subclassify the syndromes according to whether synthesis of the affected globin chain is totally absent (e.g., β0-thalassemia) or only partially reduced (e.g., β + -thalassemia).
Some mutations alter the patterns of fetal to adult Hb switching. These conditions, called hereditary persistence of fetal hemoglobin , are not generally associated with clinical symptoms; nonetheless, they merit consideration in this chapter. Their importance lies in their role as modulating factors when coinherited with other hemoglobinopathies. They are also useful models for investigating the molecular basis for globin gene regulation during human development and as paradigms for rational therapy for the major β-chain hemoglobinopathies, namely, sickle cell anemia and β-thalassemia.
Epidemiology
Thalassemias have been encountered in virtually every ethnic group and geographic location. They are most common in the Mediterranean basin and tropical or subtropical regions of Asia and Africa. The “thalassemia belt” extends along the shores of the Mediterranean, through the Arabian peninsula, Turkey, Iran, India, and southeastern Asia, especially Thailand, Cambodia, and southern China. The prevalence of carriers of thalassemia in these regions is in the range of 2.5% to 15%. Similar to sickle cell anemia, thalassemia is most common in areas historically affected by endemic malaria. Malaria seems to have conferred selective survival advantage to thalassemia heterozygotes in which infection with the malarial parasite is believed to result in milder disease and less impact on reproductive fitness. Therefore, the gene frequency for thalassemia has become fixed and high in populations exposed to malaria over many centuries. Migration from these parts to the rest of the world has resulted in the thalassemias being found all over the world, including Australia and the Americas, though β-thalassemia is still somewhat uncommon in Africa, except parts of West Africa and North Africa bordering on the Mediterranean Sea. The most severe form of α-thalassemia is found mainly in the Oriental populations because of the higher prevalence of the two gene deletions in cis configuration in these areas.
Pathophysiology: General Principles
The classification, genetic basis, and pathophysiology of the thalassemia syndromes are based on a thorough understanding of the human hemoglobins, their biosynthesis, their encoding globin gene families, and their roles as soluble oxygen-carrying molecules. Therefore, readers of this chapter should first familiarize themselves with the material presented in Chapter 34 . The material presented in this chapter is also substantially clarified by prior reading of Chapter 35, Chapter 36, Chapter 37 because the principles underlying the pathophysiology of and therapy for thalassemia draw heavily on knowledge of iron metabolism.
The primary lesion in all forms of thalassemia is reduced or absent production of one or more globin chains. Red blood cells (RBCs) contain a mix of hemoglobin types, which vary through intrauterine development. These molecules have the same heme moieties but differ in globin chains. In the post-natal period, after infancy, the predominant hemoglobin is adult hemoglobin or HbA, a heterodimer of 2 alpha and 2 beta globins (α 2 β 2 ). For all practical purposes, the major impact on clinical well-being occurs only when these lesions affect the α- or β-globin chains necessary for the synthesis of adult Hb. Severe impairment of γ-, ε-, or ζ-globin production is presumably lethal in utero, and has not been observed in human biology.
One consequence of reduced globin-chain production is immediately apparent: reduced production of functioning Hb tetramers, and consequently anemia. Thus, hypochromia and microcytosis are characteristic of virtually all patients with thalassemia. In the mildest forms of the disease, this phenomenon may be barely detectable.
The second consequence of impaired globin biosynthesis is unbalanced synthesis of the individual α- and β-subunits. Hb tetramers are highly soluble and have reversible oxygen-carrying properties exquisitely adapted for oxygen transport and delivery under physiologic conditions. Free or “unpaired” α-, β-, and γ-globin chains are either highly insoluble (“unstable”) or form homotetramers of alpha-globin (Fessas bodies), beta-globin (HbH), or gamma-globin (Hb Bart), that are incapable of releasing oxygen normally, and because they are relatively unstable, will precipitate as the cell ages. For poorly understood reasons, no compensatory regulatory mechanism exists whereby impaired synthesis of one globin subunit leads to a compensatory downward adjustment in the production of the other (partner) globin chain of the Hb tetramer. Thus, useless excess α-globin chains continue to accumulate and precipitate in red cell precursors in β-thalassemia, and excess β-globin chains form HbH in α-thalassemia. The abnormal solubility and oxygen-carrying properties of these chains lead to a variety of physiologic derangements. Indeed, in the severe forms of thalassemia, it is the behavior of the unpaired globin chains accumulating in relative excess that dominate the pathophysiology of the syndrome rather than the mere underproduction of functioning Hb tetramers. The precise complications of this pathophysiologic phenomenon are diverse and depend on the amount and the identity of the globin chain accumulating in excess.
The predominant pathophysiologic determinant in α-thalassemia is the production of HbH, with anemia and impaired oxygen carrying capacity, while in β-thalassemia it is apoptosis of the erythroid precursors in the bone marrow (BM) as a result of hemichrome formation from precipitation of alpha globin aggregates. Thus, IE, while part of both, is more severe when there is an excess of α-globin in β-thalassemia, compared with a similar degree of β-globin excess in α-thalassemia, making the pathophysiology of anemia quite different.
The predominant circulating Hb at the moment of birth is fetal hemoglobin (HbF, α 2 γ 2 ). Although the switch from γ- to β-globin biosynthesis begins before birth, the composition of Hb in the peripheral blood changes much later because of the long-life span of normal circulating RBCs. HbF is thus slowly replaced by HbA so that infants do not depend heavily on normal amounts and function of HbA until they are between 4 and 6 months old. The pathophysiologic consequences of these considerations are that whereas α-chain hemoglobinopathies tend to be symptomatic in utero and at birth, individuals with β-chain abnormalities are not very symptomatic until 4 to 6 months of age. These differences in the onset of phenotypic expression arise because α-chains are needed to form HbF and HbA, but β-chains are required only for HbA. Red cells which continue to produce gamma globin will have a selective survival advantage in β-thalassemia because they reduce the imbalance from excess α-globin.
BETA-Thalassemia Syndromes
Nomenclature
Approximately 350 mutations in the beta-globin gene cluster have been described. These mutations vary widely in severity, from complete non-production of beta-globin, called beta-zero (β 0 ), some expression and thus some production of beta-globin (β + ), to the production of abnormal beta-globins which result in abnormal hemoglobins such as HbE (β E ). There is, thus, considerable phenotypic variability in these syndromes. The most severe form was termed thalassemia major, and referred to a clinically severe disease with marked IE and its manifestations, severe anemia, lifelong transfusion dependence, complications mostly related to iron overload, and a shortened life expectancy. The genotype is typically, but not exclusively, β 0 /β 0 or β 0 /β + . At the other end of the spectrum is thalassemia minor which refers to the trait, or carrier state, with one normal β-globin gene, either β/β 0 or β/β + . In between is a broad spectrum of disease referred to as thalassemia intermedia, which is milder, with a moderate anemia which may not require regular transfusions, with more complications from IE and hemolysis than iron overload, mostly related to skeletal changes and vascular disease. Beta-globin genotypes which result in this clinical syndrome include, but are not limited to, β 0 /β + , β + /β + , β 0 /β E or β + /β E . A number of more complex genotypes may also be implicated, such as the delta-beta-thalassemias ( δβ ) + , or hemoglobin Lepore ( δβ ) 0 , or ( A γδβ ) 0 , a single β-thalassemia defect and an excess of normal alpha-globin genes, or two β-thalassemia mutations coinherited with heterozygous α-thalassemia (in this last form, known as αβ-thalassemia, the α-thalassemia allele reduces the burden of unpaired α-chains). Simple heterozygosity for certain forms of β-thalassemic hemoglobinopathies can also be associated with a thalassemia intermedia phenotype, sometimes called dominant β-thalassemia .
Terminology based on the clinical spectrum of disease previously focused on the need for regular transfusions early in life, with those individuals being classified as thalassemia major. Those who did not require regular transfusions but had other manifestations of the disease were referred to as having thalassemia intermedia. However, in the course of their disease many individuals who were initially not regularly transfused could come to need regular transfusions, and thus move along the spectrum from intermedia to major. Currently, the trend has been to simply classify patients as having transfusion-dependent thalassemia (TDT) if they are regularly transfused (requiring 8 or more transfusions per year), or non-transfusion-dependent thalassemia (NTDT) who may or may not require intermittent transfusions.
Molecular Pathology
Forms of β-thalassemia arise from mutations that affect every step in the pathway of globin gene expression: transcription, processing of the messenger ribonucleic acid (mRNA) precursor, translation of mature mRNA, and posttranslational integrity of the β-polypeptide chain ( Fig. 41.1 and Table 41.1 ). Large deletions removing two or more non– β- genes are found in rare cases, as are smaller partial or total deletions of the β -gene alone (see Fig. 41.1 ). Most types of β-thalassemia are caused by point mutations affecting one or a few bases. Of the more than 200 mutations causing β-thalassemia, approximately 15 account for the vast majority of affected patients, with the remainder responsible for the disorder in only relatively few patients. It has been determined that five or six mutations usually account for more than 90% of the cases of β-thalassemia in a given ethnic group or geographic area (see Table 41.1 ).
Table 41.1
Common β-Thalassemia Mutations in Different Racial Groups
Data from Kazazian HH Jr, Boehm CD. Molecular basis and prenatal diagnosis of beta-thalassemia. Blood . 1988;72(4):1107. https://pubmed.ncbi.nlm.nih.gov/23637309/
| Racial Group | Description |
|---|---|
| Mediterranean | IVS-1, position 110 (G → A) |
| Codon 39, nonsense (CAG → TAG) | |
| IVS-1, position 1 (G → A) | |
| IVS-2, position 745 (C → G) | |
| IVS-1, position 6 (T → C) | |
| IVS-2, position 1 (G → A) | |
| African | −34 (A → G) |
| −88 (C → T) | |
| Poly(A), (AATAAA → AACAAA) | |
| Southeast Asian | Codons 41/42, frameshift (-CTTT) |
| IVS-2, position 654 (C → T) | |
| −28 (A → T) | |
| Asian Indian | IVS-1, position 5 (G → C) |
| 619-bp deletion | |
| Codons 8/9, frameshift (++G) | |
| Codons 41/42, frameshift (−CTTT) | |
| IVS-1, position 1 (G → T) |
Several mutations alter the promoter region upstream of the β-globin mRNA-encoding sequence, impairing mRNA synthesis. Mutations in the upstream “master switch” or “LCR” sequences that are needed to induce the high rate of β-globin expression also lead to inadequate production of globin mRNA, while mutations that derange the sequence used as the signal for the addition of the poly-(A) tail of the mRNA polyadenylation signal have been shown to result in abnormal cleavage and polyadenylation of the nascent mRNA precursor, with resulting reduced accumulation of mature mRNA.
Processing
Many forms of β-thalassemia are caused by mutations that impair splicing of the mRNA precursor into mature mRNA in the nucleus or that prevent translation of the mRNA in the cytoplasm. The molecular pathology of splicing mutations is complex ( Fig. 41.2 ). Some base substitutions ablate the donor (GT) or acceptor (AG) dinucleotides, which are absolutely required at the intron–exon boundaries for normal splicing and thereby completely block production of mature functional messenger RNA. Thus, no β-globin can be synthesized (β0-thalassemia). Other mutations alter the consensus sequences that surround the GT- and AG-invariant dinucleotides and decrease the efficiency of normal splicing signals by 70% to 95%, resulting in β + -thalassemia; some consensus mutations even abolish splicing completely, causing β°-thalassemia. A third type of splicing aberration results from mutations that are not in the immediate vicinity of a normal splice site. These alter regions within the gene, called cryptic splice sites , which resemble consensus splicing sites but do not normally sustain splicing (see Fig. 41.2 ). The mutations activate the site by supplying a critical GT or AG nucleotide or by creating a sufficiently strong consensus signal to stimulate splicing at that site 60% to 100% of the time. The activated cryptic sites usually generate an abnormally spliced, untranslatable mRNA species. Only 10% to 40% of the mRNA precursors are thus spliced at the normal sites, which causes β + -thalassemia of variable severity. The mutation responsible for the most common form of β-thalassemia among Greeks and Cypriots ( Fig. 41.3 ) activates a cryptic splice site near the 3′ end of the first intron (position 110). The determinants that dictate the degree to which each mutation alters splice site use remain largely unknown.
Translation
Mutations that abolish translation occur at several locations along the mature mRNA and are very common causes of β-thalassemia (see Fig. 41.1 and Table 41.1 ). The most common form of β°-thalassemia in Sardinians results from a base substitution in the gene that changes the codon encoding the 39th amino acid of the β-globin chain from CAG, which encodes glutamine to TAG, whose equivalent (UAG) in mRNA specifies termination of translation (see Fig. 41.3 ). A premature termination codon totally abrogates the ability of the mRNA to be translated into normal β-globin. Premature translation termination also results indirectly from frameshift mutations (i.e., small insertions or deletions of a few bases, other than multiples of three, that alter the phase or frame in which the nucleotide sequence is read during translation). An in-phase premature termination codon is usually encountered within the next 50 bases downstream from a frameshift.
Other Sites
Rare mutations that affect gene function by intriguing mechanisms have been described. An extremely large deletion of the β-globin gene cluster has been described that removes the ε-, γ-, and δ-genes. The patient has a severe β-thalassemia phenotype, but the β-globin gene and 500 bases of adjacent 5′ and 3′ DNA have an entirely normal nucleotide sequence. The β-gene functions normally in surrogate cells. The important aspect of this deletion is that it removes the critical locus control region located thousands of bases upstream from the beginning of the globin gene cluster at the 5′ end of the ε-globin gene; loss of this region severely impairs β-gene expression. A number of additional deletions involving the locus control region and various portions of the β-gene cluster, but sparing the β-gene itself, have the same phenotype.
In other cases of β-thalassemia, the β-gene and adjacent DNA are structurally normal, and the basis of abnormal gene expression is unknown.
Relationship Between Specific Mutations and Clinical Severity
The relationship between an individual mutation and the clinical severity of the β-thalassemia phenotype associated with that particular mutation is complex. For example, the A to G mutation at position 34 of the β-gene promoter commonly encountered in patients of African origin is associated with a different clinical severity than that found in Chinese patients inheriting the same mutation. Clearly, the genetic “context” of the mutation is different in the two populations. The mutant β-globin gene in the two different racial groups probably arose in different chromosome backgrounds that have different potentials for γ-gene expression. Multiple forms, or haplotypes, of normal non–α-globin gene clusters exist in various human populations. These were defined by the patterns of restriction fragment length polymorphisms detected when DNA is digested with restriction endonucleases and analyzed by Southern gene blotting for the fragments bearing the non–α-globin genes. The gene cluster is now readily analyzed by direct genomic sequencing. Haplotypes differ according to whether each restriction site is present or absent along the gene cluster. More than 12 haplotypes have been defined by examination of several restriction sites located along the cluster that are present or absent in a polymorphic manner in normal individuals. The clinical variability encountered in two different groups bearing identical primary mutations correlates best with the haplotype or chromosome background on which the mutation is inherited. The differences in physiologically important functions among haplotypes that modulate severity remain unknown, but a possible explanation lies in the variable abilities of the γ-globin genes on different chromosomes to respond to severe erythroid stress by increased expression during postnatal life. The β-globin genes carried on some haplotypes differ in the degree to which they can respond in this manner. Because HbF synthesis reduces the severity of β-chain hemoglobinopathies, the level of γ-gene expression from a given chromosome can play an important modulating role.
Pathophysiology
Erythropoiesis is essentially a two-phase process, erythropoietin (EPO) driving the initial proliferative phase, leading to stimulation of the stem cell toward the erythroid lineage, and proliferation through the BFU-E and CFU-E phases. The second stage is one of differentiation and maturation, with the proerythroblasts moving through the different erythroblast phases, into reticulocytes and eventually mature red cells ( Fig. 41.4 ).
As described, the basic molecular abnormality in β-thalassemia is the imbalance between alpha and beta globin production, with reduction in beta globin production and thus a decrease in HbA (α 2 β 2 ) production. The ratio of β- to α-globin is typically close to 1. When abnormal, this ratio has a direct correlation with clinical severity in β-thalassemia patients. In β-thalassemia heterozygotes, β-globin synthesis is approximately half-normal, with the ratio of β- to α-chain mRNA (β/α ratio) of 0.5 to 0.7 (normal = 1.0). In homozygotes for β0-thalassemia, which account for approximately one-third of patients, β-globin synthesis is absent. β-globin synthesis is reduced to 5% to 30% of normal levels in β + -thalassemia homozygotes or β + /β°-thalassemia compound heterozygotes, which together account for approximately two-thirds of cases. The excess α-globin chains may be stabilized by alpha Hb stabilizing protein, which is a chaperone-like protein that assists in binding free α-globin chains. Higher levels of alpha Hb stabilizing protein result in more IE, and thus a more severe clinical phenotype.
The decreased or absent synthesis of HbA (α 2 β 2 ) results in the production of smaller red cells and depending on the severity of the deficiency, hypochromia as well. RBCs are always microcytic in the thalassemias across the spectrum, and usually hypochromic though not so much in heterozygotes. γ-Chain synthesis is partially reactivated, and there is some upregulation of δ chain synthesis, though not enough to replace the deficiency of β-chains, and thus the red cells may contain relatively large proportions of HbF (α 2 γ 2 ) and some HbA2 (α 2 δ 2 ).
Individuals inheriting two β-thalassemic alleles experience a more profound deficit of β-chain production. Little or no HbA is produced, and importantly, the imbalance of α- and β-globin production is far more severe ( Fig. 41.5 ). In the BM, the limited capacity of the red cell precursors to proteolyze the excess α-globin chains, a capacity that probably exerts a protective effect in heterozygous β-thalassemia, is overwhelmed in homozygotes. Free α-globin accumulates, and unpaired α-chains aggregate and precipitate to form inclusions called Fessas bodies and hemichromes with the addition of heme iron, which cause oxidative membrane damage leading to apoptosis and destruction of immature developing erythroblasts in the BM (intramedullary hemolysis). Consequently, relatively few of the erythroid precursors undergoing erythroid maturation in the BM survive long enough to be released into the bloodstream as erythrocytes, leading to anemia. Thus, in β-thalassemia, the defect in the erythropoietic pathway is one of impaired differentiation and maturation of the developing erythroid precursors, beginning at the time globin genes begin expression in the proerythroblast-basophilic erythroblast stages. Erythropoiesis is ineffective , and the severity of this IE worsens as the imbalance between β- and α-globin synthesis deepens. The occasional erythrocytes that are formed during erythropoiesis bear a burden of inclusion bodies. The reticuloendothelial cells in the spleen, liver, and BM remove these abnormal cells prematurely, which reduces RBC survival as a consequence of this hemolytic anemia.
IE is thus the hallmark of β-thalassemia, triggering a cascade of compensatory mechanisms and resulting in clinical sequelae such as erythroid BM expansion, extramedullary hematopoiesis (EMH), and splenomegaly. The master iron regulatory hormone hepcidin plays an integral and critical role in the pathophysiology of the disease as well (see Chapter 35, Chapter 36, Chapter 37 ). Erythroferrone (ERFE) is a protein which is produced during erythropoiesis, which has the effect of reducing hepcidin production, likely through the BMP/SMAD signaling pathway. During stress erythropoiesis, increased ERFE results in decreased hepcidin which in turn leads to increased iron absorption through the transporter ferroportin at the basolateral side of the intestinal enterocyte, the body mistakenly sensing anemia as being a result of iron deficiency. The greater the erythropoietic activity, the higher ERFE levels rise to, and the greater the suppression of hepcidin. In thalassemia, where IE is a significant feature, ERFE levels are high and therefore hepcidin levels are inappropriately low. Such patients become iron overloaded as a result of increased iron absorption even in the absence of transfusional iron loading. The critical role of hepcidin in iron trafficking is also a factor in iron toxicity in thalassemia. In the absence of IE, hepcidin levels are increased when there is iron overload, thus reducing iron absorption, and also safely sequestering iron in storage sites and preventing deposition in vulnerable tissues. The relatively lower levels of hepcidin driven by ERFE in ineffective erythropoiesis result in iron being able to be more easily redistributed, leading to organ toxicity. When hepcidin production is inappropriately low as a result of increased IE, tissues such as the heart and endocrine organs are exposed to more circulating iron and this results in uptake and organ dysfunction as a result of iron toxicity.
As a result of the anemia, there is increased secretion of EPO, and further proliferation, once again, without maturation. Accumulation of molecules belonging to the transforming growth factor-beta (TGF-β) superfamily of ligands has been described, which inhibits maturation and differentiation. Trapping these ligands has been the focus of a therapeutic intervention to try and improve IE by removing the block on maturation, and promoting more effective erythropoiesis. Massive BM expansion does occur, but very few erythrocytes are actually supplied to the circulation. The BM becomes packed with immature erythroid precursors, which die from their burden of precipitated α-globin chains before they reach the reticulocyte stage. Profound anemia persists, driving erythroid hyperplasia to still higher levels. In some cases, erythropoiesis is so exuberant that masses of extramedullary erythropoietic tissue form in the chest, abdomen, or pelvis.
Clinical Manifestations
Anemia and IE are central to the pathophysiology and disease manifestations of thalassemia, and the clinical manifestations are a cascade that results from these ( Fig. 41.6 ). However, there is tremendous heterogeneity in the spectrum of clinical features in the various thalassemia syndromes, determined by the genotype, genetic modifiers, age at presentation, and diagnostic and treatment modalities employed. Two siblings inheriting identical thalassemia mutations sometimes exhibit markedly different degrees of anemia and erythroid hyperplasia. Many factors contribute to this clinical heterogeneity. Individual alleles vary with respect to severity of the biosynthetic lesion. Other modifying factors ameliorate the burden of unpaired α-globin. Levels of γ-globin expression are widely variable in β-thalassemia, with greater production of γ-globin able to reduce the excess of α-globin. Theoretically, patients may also vary in their ability to solubilize unpaired globin chains by proteolysis. Occasional heterozygous patients have had more severe anemia than expected, possibly because of defects in these proteolytic systems or because of the type of thalassemic mutation. Inheritance of more than the usual complement of α-globin genes may also increase with severity of β-thalassemia because of additional production of unpaired α-globin chains. All of these factors emphasize the essential role of α-globin inclusions in the pathophysiology of β-thalassemia.
In general, in patients who have not been transfused regularly, manifestations are mainly from the effects of IE, with bone changes and vasculopathy dominating, but iron overload remaining an important feature as well. In appropriately transfused individuals, the effects of the now suppressed IE are not prominent, and it is more the effects of iron overload that present as complications.
Beta-Thalassemia Major
Homozygotes or compound heterozygotes with the more severe genotypes are born, usually without prenatal complications, with mild anemia and microcytosis compared with newborns without these mutations. Nevertheless, deficient β-chain synthesis can be demonstrated at birth on the newborn screen, which would show an absence or marked reduction in HbA. As the Hb declines to the physiologic nadir, which is reached sooner as a result of the relative anemia at birth, erythropoiesis resumes under the drive of EPO. However, since the marrow is not able to produce adequate red cells, the Hb level continues to drop leading to further EPO production and BM hyperplasia, and persistent red marrow in bones which would otherwise have converted to yellow marrow. In addition, normally quiescent erythroid precursors in the liver and spleen are activated, with resulting EMH. Clinical manifestations usually emerge as the anemia becomes more pronounced. The diagnosis is almost always evident by 2 years of age. Pallor, irritability, failure to thrive, growth retardation, abdominal swelling caused by enlargement of the liver and spleen, and jaundice are the usual presenting features. Facial and skeletal changes caused by BM expansion develop as the anemia worsens, with frontal and parietal bossing of the skull bones, persistent marrow in the maxilla resulting in underdevelopment of normal sinuses ( Fig. 41.7 ).
Untreated patients die in late infancy or early childhood as a consequence of severe anemia. In a retrospective review from Italy, the average survival of children with untreated thalassemia major was less than 4 years; approximately 80% died in the first 5 years of life. Patients who receive transfusions sporadically may live somewhat longer than untransfused patients, but their quality of life is extremely poor as a result of both the chronic anemia and the IE. The low Hb level and massive organomegaly are usually disabling, and the changes in the facial bones are disfiguring. After 10 to 20 years of weakness, stunted growth, and impaired activity, the undertransfused patients usually succumb to congestive heart failure.
This disastrous symptom constellation, so prevalent in the past, is now rare in most industrialized countries. Nonetheless, the clinical manifestations and complications of untreated or undertreated β-thalassemia major illustrate the principles of the pathophysiology. Furthermore, these descriptions accurately characterize the disease that is still prevalent in many underserved parts of the world.
Beta-Thalassemia Intermedia
Individuals with less severe genotypes have a milder course than that described above, but always have some pathologic features of the disease unlike heterozygotes who are mostly completely asymptomatic. However, this intermediate syndrome has a wide spectrum of severity based on the degree of IE and its consequences, and thus individuals may have mild symptoms to more complex presentations which may move them along the spectrum toward transfusion dependence. Approximately 10% of patients with homozygous β-thalassemia may exhibit a phenotype characterized by intermediate hematologic severity. The balance of globin chain synthesis is better than in typical thalassemia major because of a less severe defect in β-globin chain synthesis, a decrease in α-globin chain synthesis along with decreased β-globin chain synthesis, or an increase in γ-globin chain synthesis. For example, homozygous β-thalassemia in African Americans, Portuguese, and other populations may be relatively mild, at least for the first two decades of life. Homozygotes or mixed heterozygotes for forms of β-thalassemia associated with normal HbA 2 and normal HbF (so called “silent carrier” mutations) also tend to have mild to moderate disease.
Unfortunately, many individuals with milder presentations are not appropriately diagnosed, being treated as chronic or refractory iron deficiency instead. More severely affected individuals have more marked IE, bone changes, and more severe anemia, with fatigue, abnormal bones of the skull, hepatosplenomegaly, and pathologic fractures. Individuals may present at a variable age, some in the first decade of life, some in the second, and some as late as the third. Patients do not remain static on the spectrum from thalassemia intermedia to thalassemia major. Complications in these individuals are primarily related to IE, and depending on the severity of the IE, may be mild or more severe. These complications will be described later in this chapter. Those with severe IE and anemia would likely benefit from regular transfusion. However, many such individuals either refuse regular transfusions for fear of transfusion-related infections or iron overload, or are not able to get them for logistic and economic reasons. These individuals may have growth retardation, chronic fatigue and malaise, bone pain, and worsening splenomegaly and still develop iron overload. Splenectomy is often performed when there is a component of hypersplenism contributing to worsening anemia. Gallbladder disease from increased red cell turnover and the development of pigment gallstones is quite common. Progressive EMH may result in paraspinal nodules as described previously. Particularly in splenectomized patients, vascular damage from fragmented and abnormal red cells, complicated by thrombocytosis may lead to clinical manifestations including the development of chronic, non-healing leg ulcers from poor perfusion and the effects of arterial endothelial damage and a possible hypercoagulable state leading to cerebral infarcts and development of pulmonary hypertension. Some individuals may remain in the intermedia spectrum until adulthood, receiving very few transfusions for stress situations such as severe infections or surgery, when they may have a drop in Hb and worsening symptoms related to anemia.
Clinically significant iron loading as a result of increased absorption is seen even in patients with infrequent transfusions (see Chapters 35 and 36 ). Iron overload may result in diabetes and endocrine disturbances, typically by fourth decade of life, though cardiac deposition is relatively rare in true thalassemia intermedia individuals.
Individuals with the most severe form of the disease will usually be placed on a regimen of regular transfusions. In appropriately transfused individuals who have suppressed IE, complications are now mostly related to those resulting from transfusional iron overload.
Beta-Thalassemia Minor
Heterozygotes for β-thalassemia are usually asymptomatic. Often the diagnosis is made as a result of their family history, a prenatal screen, as part of the workup for refractory iron deficiency anemia, or by the chance finding of the characteristic hematologic changes during a routine study. In general, thalassemia trait carries no direct clinical symptoms or pathologic consequences for the patient. Usually, they have adapted to a mild microcytic anemia, with no symptoms, no overt evidence of IE or EMH. A controlled study reported that individuals with the β-thalassemia trait suffer from fatigue and other symptoms indistinguishable from those with mild anemias from other causes. There was no difference in the frequency of palpable splenomegaly between the thalassemic and control groups. There are some reports in the literature of individuals who have symptoms from anemia and may even have mild splenomegaly, but in the absence of genetic testing, it is not clear that these were all heterozygotes only. In situations in which there is an adaptive increase in Hb, such as endurance activities like running marathons, or living at high altitude, individuals may have some difficulty. The diagnosis of thalassemia trait assumes particular importance in women who are pregnant or considering pregnancy because of the potential for having a child with thalassemia major. During pregnancy, the anemia of thalassemia trait often becomes more severe, but transfusions are rarely necessary. Increased folic acid supplementation may improve Hb during this period. Because iron deficiency may occur during pregnancy, iron supplementation has been advised to avoid compounding the causes of anemia. Studies have suggested there may be an increased tendency for gallstones and cholecystitis, but otherwise this condition should be largely asymptomatic. Some β-thalassemia carriers have increased iron stores, although this is most often a result of inappropriate long-term iron therapy based on a misdiagnosis. In countries where there is a relatively high frequency of genetic determinants for hemochromatosis, the possibility of their coinheritance should be borne in mind if a patient with β-thalassemia trait with an unusually high plasma iron or serum ferritin level is encountered. Iron supplementation is only necessary if there is true iron deficiency, and routine supplementation to raise the Hb level is futile and should not be done. These individuals should seek genetic counseling when ready for childbearing.
Laboratory Findings
In order to make the diagnosis, the clinician must consider the ethnicity, family history, clinical presentation, physical exam as well as laboratory parameters. The laboratory diagnosis of the thalassemia syndromes has traditionally been based on careful interpretation of the complete blood count, including the red cell indices, the review of the peripheral blood smear, and characterization of the type of Hb produced. Hb levels vary based on the severity of the underlying genotype, but the anemia is always microcytic with low red cell indices. The smear shows variable hypochromia and the presence of abnormal shapes and sizes of cells, all more so in individuals with increasing severity of anemia. There is a reduction or absence of production of HbA, and thus an increase in HbF and HbA2 on Hb fractionation. Special staining, α-, β-, and γ-globin assessments are performed in some sophisticated laboratories, along with some degree of genotyping, especially for known mutations. In the developed world, it is now possible to characterize the disease more completely, including all of the possible genetic modifiers with the easy availability of sophisticated DNA testing, including whole exome or genome sequencing. While this is not routinely done for all patients, especially those with mild clinical manifestations, in more complex cases, it may be used to fully explain the clinical picture. There may also be some predictive value in genotyping for some of the newer therapies, as will be described later. Family members should also have testing if they have not been tested previously.
The diagnosis is usually fairly easy to make, though the following may be considered if it is not immediately clear:
-
congenital sideroblastic anemia, one of the rarer instances when a BM exam or specific genetic testing may be needed to make the diagnosis
-
juvenile chronic myelogenous leukemia may be superficially considered, because of the elevated HbF levels, but other testing is quite straightforward to make this diagnosis
Homozygous β 0 Thalassemia
Infants are born with relative anemia and microcytosis. The Hb level decreases progressively during the first months of life. When the child becomes symptomatic, the Hb level may be as low as 3 to 4 g/dL. RBC morphology is strikingly abnormal, with many microcytes, bizarre poikilocytes, teardrop cells, and target cells ( Fig. 41.8 ). A characteristic finding is the presence of extraordinarily hypochromic, often wrinkled and folded cells (leptocytes) containing irregular inclusion bodies of precipitated α-globin chains. Nucleated RBCs are frequently present in peripheral circulation. The reticulocyte count is 2% to 8%, lower than would be expected in view of the extreme erythroid hyperplasia and hemolysis. The low count reflects the severity of intramedullary erythroblast destruction. The white blood cell count is elevated. A moderate polymorphonuclear leukocytosis and normal platelet count are typical unless hypersplenism has developed. The BM (not necessary for diagnosis) exhibits marked hypercellularity caused by erythroid hyperplasia. The RBC precursors show a maturational arrest with defective hemoglobinization and reduced amounts of cytoplasm.
The osmotic fragility is strikingly abnormal. The RBCs are so markedly resistant to hemolysis in hypotonic sodium chloride solution that some are not entirely hemolyzed even in distilled water. Even before transfusion therapy is initiated, the serum iron and transferrin saturation and serum ferritin may already be increased as a result of increased iron absorption, thus ruling out iron deficiency as an alternate diagnosis.
The Hb profile reveals predominantly HbF. In patients with homozygous β0-thalassemia, HbA is absent throughout life, unless transfused. In β + -thalassemia HbA may be very low or undetectable in the newborn but present in reduced amounts in later life. The levels of HbA2 in thalassemia major are variable, probably because of increased numbers of F cells that have a decreased HbA2 content. Other biochemical abnormalities of the RBC in cases of thalassemia major include a postnatal persistence of the RBC in antigen and a decrease of RBC carbonic anhydrase; these findings are probably also caused by the elevated levels of circulating F cells.
The intraerythrocytic inclusions in the peripheral blood cells of patients with thalassemia, termed Fessas bodies, are especially prominent after splenectomy. These inclusions, best seen by staining with supravital staining (Brilliant Cresyl Blue) or by phase microscopy, are aggregates of precipitated, denatured α-chains. They are also found in large numbers within erythroid precursors in the BM.
RBC survival in cases of thalassemia major is variable but usually markedly decreased. The 53 Cr half-life ranges between 6.5 and 19.5 days compared with the normal half-life of 25 to 35 days, but there is some variability with cells containing more fetal Hb surviving longer. Increased plasma iron turnover and poor use of radiolabeled iron provide further evidence of IE.
Unconjugated bilirubin levels are elevated, in the range of 2.0 to 4.0 mg/dL at the time of diagnosis but may rise substantially as the anemia worsens in the absence of transfusion. Serum aspartate aminotransferase levels are frequently increased at diagnosis because of hemolysis. Alanine aminotransferase levels are usually normal before transfusion therapy but may rise subsequently because of iron-induced hepatic damage or viral hepatitis. Lactate dehydrogenase levels are markedly elevated as a consequence of IE. Haptoglobin and hemopexin are reduced or absent.
Hypersplenic patients may have low white cell and platelet counts, and after splenectomy, leukocytosis and thrombocytosis are common. Asplenic individuals usually have large, flat “pseudo-macrocytes,” or leptocytes that appear larger in two dimensions because they are so devoid of Hb that they collapse flat on the slide like a deflated balloon, and small, deformed microcytes, as well as markedly increased nucleated red cell counts, and the white cell count frequently has to be corrected for their presence. Staining of the blood with methyl violet, particularly in splenectomized subjects, reveals stippling or ragged inclusion bodies in the red cells.
When transfusions are initiated, the Hb level rises to the mild to moderate anemia range, being higher in patients on regular transfusions and generally lower in those receiving on-demand transfusions. The mean corpuscular volume (MCV) and Hb fractionation reflects transfused blood as well. Iron parameters are altered to reflect iron overload as transfusions continue. Serum iron and transferrin saturation, as well as ferritin levels are elevated, and remain so, with the ferritin level declining in response to chelation therapy.
Transfusion complications include the development of auto- or alloantibodies with a positive DAT and antibody screen, iron-related organ dysfunction, hepatic, and endocrine. All of these parameters must be regularly monitored in transfusion-dependent patients.
Homozygous or Compound Heterozygous β + Thalassemia
In these individuals, the Hb levels are generally higher, but otherwise the hematologic changes are similar. The more severe the anemia, with greater IE, the more the peripheral smear resembles that of a thalassemia major patient prior to starting transfusions, as noted above. Examination of the BM, if performed, would confirm IE to a variable degree, with more severe patients having the picture of untreated thalassemia major. Hb fractionation shows a variable amount of HbA depending on the specific genotype, and the rest is HbF and A2, or in cases of HbE-thalassemia, a proportion of HbE. Even in the absence of transfusions, iron studies will show a steady rise in serum iron, transferrin saturation, and ferritin as a result of increased intestinal absorption, the rate of rise determined by the degree of IE. With intermittent or regular transfusions, the iron levels will rise more rapidly, and follow the same course as individuals with thalassemia major.
Heterozygous β-Thalassemia
Inheritance of a single β-thalassemia allele usually results in a mild anemia. The classic picture of β-thalassemia minor is a mild microcytic anemia (Hb averages 1 or 2 g/dL lower than that seen in normal persons of the same age and gender, typically 9 to 11 g/dL, MCV 50 to 70 fL). The red cell count is usually normal or elevated. HbF levels decline more slowly than usual in the first year of life, and the diagnostic elevated HbA2 levels are established by approximately 6 months of age. Strong intrafamilial correlations of both HbA2 and MCV are noted. The HbA2 level is increased to 3.5% to 7%. The level of fetal Hb is elevated in approximately 50% of cases, usually to 1% to 3% and rarely to greater than 5%. The RBC count is increased or normal. The RBCs are characteristically hypochromic (mean corpuscular hemoglobin [MCH] <26 pg). Osmotic fragility is decreased; indeed, a one-tube osmotic fragility test has been used in the past for mass screening. The smear shows varying numbers of target cells, poikilocytes, ovalocytes, and basophilic stippling ( Fig. 41.9 ). The reticulocyte count is normal or slightly elevated. RBC survival is normal, iron utilization is decreased, and slight IE is present. Iron studies are normal, unless concomitant iron deficiency is also present, in which case the transferrin saturation and ferritin are low. In this situation, the Hb is lower, as are the red cell indices, and the red cell count. The BM (not usually done) in heterozygous β-thalassemia shows slight erythroid hyperplasia with rare red cell inclusions. Megaloblastic transformation as a result of folic acid deficiency occurs occasionally, particularly during pregnancy. A mild degree of IE is noted, but red cell survival is normal or nearly normal.
β-Thalassemia With Normal A2 Level
Rare forms of β-thalassemia are seen in which heterozygotes have a completely normal hemogram, or with mild microcytic anemia but with a completely normal Hb pattern on electrophoresis or high-pressure liquid chromatography (HPLC). This can be confused with the more common heterozygous α-thalassemia and may cause difficulties in genetic counseling and prenatal diagnosis. Based on hematologic studies, two main classes of “normal HbA2 β-thalassemia”—sometimes called types 1 and 2—are seen.
Type 1, the “silent” form of β-thalassemia, is characterized in heterozygotes by a normal hemogram—there is no anemia, and the MCV is normal. Although this condition can be partly identified by demonstrating a mild degree of globin-chain imbalance, with α-to-β synthesis ratios of approximately 1.5:1, it can only be diagnosed with certainty by DNA analysis. Compound heterozygotes for this condition and β0-thalassemia have a mild form of β-thalassemia intermedia.
Normal HbA2 β-thalassemia type 2 in heterozygotes has a hematologic profile of mild microcytic anemia which is indistinguishable from typical β-thalassemia with elevated HbA2 levels. The homozygous state has not been described. The compound heterozygous state for this gene and for β-thalassemia with raised HbA2 levels is characterized by a clinical picture of severe transfusion-dependent β-thalassemia.
Dominant β-Thalassemia
The clinical features of the dominant β-thalassemias resemble the features of thalassemia intermedia. The blood picture shows the usual thalassemia changes, and there is moderate anemia and splenomegaly. The marrow shows erythroid hyperplasia with well-marked inclusion bodies in the red cell precursors, which may be seen in the peripheral blood after splenectomy. Hb analysis shows HbA and elevated HbA2, but the HbF level is not usually elevated much higher than that seen in β-thalassemia heterozygotes.
You're Reading a Preview
Become a Clinical Tree membership for Full access and enjoy Unlimited articles
If you are a member. Log in here