CHAPTER 8: Thalassemia

The thalassemias are an inherited group of disorders in which mutations in genes expressing alpha globin or beta globin result in impaired hemoglobin synthesis and microcytic anemia of varying severity. The thalassemias are subdivided into alpha (α) or beta (β) according to which globin genes are defective. Heterozygotes are generally asymptomatic, whereas individuals who inherit thalassemia genes from each parent often have life-threatening clinical manifestations. The thalassemias have attracted worldwide interest and attention because of their high prevalence and clinical importance. Moreover, an ever-expanding body of information on the molecular pathogenesis of the thalassemias has provided critical insights into fundamental problems in biology, particularly tissue-specific and development-specific gene regulation.

A diagram of the layout of the human globin genes is shown in Figure 8-1. A tandem pair of α-globin genes is located on chromosome 16, downstream from two embryonic α-like genes called zeta (ζ). The high homology of the α-globin genes leads to frequent unequal meiotic crossover events, which are the basis for the deletions that cause the α-thalassemias, discussed at the end of this chapter.

The β-globin gene is a member of a family located on chromosome 11. As in the α-globin gene family, epsilon (∊), the most 5′ (upstream) of these genes, is expressed only in early embryos. Downstream from this gene are two tandem gamma (γ) genes whose product is found in fetal hemoglobin (Hb F, α₂β₂), the hemoglobin that predominates throughout most of gestation. The delta (δ) gene product forms a minor hemoglobin component, Hb A₂ (α₂β;₂), which has no functional importance but is useful in the diagnosis of the thalassemias (discussed later in this chapter.) The most 3′ (downstream) member of the family is the β gene whose product combines with α-globin to form Hb A (α₂β₂), the major hemoglobin component of adult red cells.

Figure 8-2 shows the expression of globin genes during development. Throughout gestation there is coordinate synthesis of globin products from the two chromosomes, permitting the sequential and orderly production of functional tetrameric hemoglobins. In the transition from embryo to fetus to birth, the globin genes on both chromosomes are "read" sequentially from left to right (5′ to 3′). During the first month of gestation, embryonic hemoglobins (ζ₂∊₂, ζ₂γ₂, ζ₂γ₂) are formed in erythroid cells located primarily in the yolk sac. During the remainder of fetal life, the sites of erythropoiesis gradually shift from the liver and spleen to the bone marrow. Fetal red cells contain predominantly Hb F (α₂γ₂). Shortly before birth, there is a dramatic switch from γ-globin to β-globin expression, which is complete by the time an infant is 6 to 8 months old. From then on, over 95% of the hemoglobin in normal red cells is adult Hb A (α₂β₂). The remaining hemoglobin consists of two minor components, Hb A₂ and Hb F. The molecular basis of the switch from γ- to β-globin synthesis remains one of the foremost problems in hematology research and, as discussed later, impacts importantly the pathogenesis and treatment of β-thalassemia as well as of sickle cell disease (Chapter 9).

During their maturation, erythroid cells become increasingly dedicated to the production of hemoglobin. Far upstream of the globin genes are regulatory elements called locus control regions at which erythroid-specific transcription factors bind cooperatively to response elements in the DNA to assure tissue-specific, high-level, and closely matched transcription of α and β (or γ) globin ribonucleic acids (RNAs). Like other precursor RNAs, globin RNA must be processed into messenger RNA (mRNA) and transported from the nucleus to cytoplasm for translation (protein synthesis) to occur. All globin genes have three exons and two introns. As shown in Figure 8-3, precursor RNA processing involves sequential splicing of the two introns followed by capping at the 5′ end of the RNA, which greatly enhances translation efficiency, along with cleavage and polyadenylation of the 3′ untranslated region, which enhances mRNA stability. Mutations at critical sites involved in splicing, capping, and polyadenylation can result in defective globin synthesis and therefore thalassemia.

As erythroid precursors reach full maturation, over 95% of protein synthesis is dedicated to globin production. This remarkable up-regulation is matched by markedly enhanced synthesis of heme. In erythroblasts and reticulocytes, globin released from the polyribosome combines with heme to form subunits capable of assembly into heterotetramers such as Hb A (α₂β₂). This assembly depends on balanced synthesis of α and β subunits. As shown in Figure 8-4, globin chain synthesis is imbalanced in the thalassemias. The presence of excess free globin chains is an important contributor to the pathogenesis of these disorders.

As shown in Figure 8-1, chromosome 11 contains a single β-globin gene. Individuals who inherit a β-thalassemia mutation from one parent and a normal β-globin gene from the other are heterozygotes, often designated as having β-thalassemia trait or β-thalassemia minor. Individuals who inherit the same β-thalassemia mutation from each parent are true homozygotes. Because multiple types of mutations can give rise to β-thalassemia, an individual often inherits a different mutation from each parent and is therefore a compound heterozygote. If homozygotes or compound heterozygotes have severe disease, they are designated as having β-thalassemia major or Cooley anemia, whereas if they have milder clinical manifestations, they have β-thalassemia intermedia.

Over 100 million people in the world are heterozygous carriers of a β-thalassemia mutation, nearly two-thirds from Asia and the rest divided between Africa, Europe, and the Americas. Affected individuals come primarily from tropical areas. Reasonably convincing evidence suggests that β-thalassemia heterozygotes are protected against infantile falciparum malaria, which is often fatal. Thus natural selection has enabled β-thalassemia genes to gradually rise to high levels in populations where malaria is endemic.

GENE DEFECTS CAUSING β-THALASSEMIA

The β-thalassemias are caused by diverse mutations involving the promoter, coding sequences, intron-exon boundaries, and the polyadenylation site of the β-globin gene. The mutant alleles are conveniently subdivided into two groups: β⁰, in which no β-globin is detectable, and β⁺, in which a small amount of normal β-globin protein is produced. Most β⁰-thalassemia alleles involve single base substitutions in the coding region of the β polypeptide that introduce premature stop codons or small insertions or deletions that cause shifts in the reading frame of the mRNA. In either case, the resulting truncated polypeptide is dysfunctional and so unstable that it is undetectable in the patient's red cells. Other less common genetic mechanisms that result in β⁰ alleles include deletions and mutations at splice junctions.

In β⁺-thalassemia, the defective gene permits the production of normal β-globin, but the amount is markedly reduced. This type of β-thalassemia is usually due to a single base substitution that either creates a new (false) splice site or lowers the efficiency of a normal splice site. In both instances, some normal splicing occurs, and some normal β-globin is made, albeit in decreased amounts. When the alternate splice site is used, a nonsense mRNA is produced that cannot form a stable or useful protein product. Less often β⁺-thalassemia is caused by mutations in the 5′ promoter or in a 3′ site in β-globin RNA where it is cleaved prior to polyadenylation.

CELLULAR PATHOGENESIS OF β-THALASSEMIA

Because inadequate amounts of β-globin are produced, there is a deficiency in the amount of hemoglobin per red cell. Therefore, the mean cell hemoglobin concentration and the mean cell volume (MCV) are decreased. Red cell production is impaired because of intramedullary destruction of erythroid precursors (ineffective erythropoiesis, Chapter 3). In addition, the survival of circulating red cells is somewhat decreased. These abnormalities are present to a slight degree in heterozygous carriers and to a marked degree in homozygotes or compound heterozygotes. In severely anemic patients, increased erythropoietin production markedly stimulates erythropoiesis in marrow cavities of all the bones as well as in extramedullary sites such as the liver and spleen, resulting in enlargement of these organs.

The markedly enhanced destruction of erythroid cells in these patients can be explained by chain imbalance. As shown in Figure 8-4, the β/α synthesis ratio is about 0.5 in heterozygotes and only 0.1 in homozygotes or compound heterozygotes. In the latter individuals, α-globin subunits are present in huge excess. Some, as depicted in Figure 8-5, partner with γ-globin to form Hb F, the dominant hemoglobin in red cells of patients with the severe types of β-thalassemia. The remaining free α-globin is poorly soluble and forms a precipitate in erythroid precursor cells. Its heme groups auto-oxidize, releasing toxic reactive oxygen species, that, as depicted in Figure 8-6, damage the erythroid cell membrane, leading to recognition and destruction by macrophages in the marrow, liver, and spleen. This severely ineffective erythropoiesis greatly impairs red cell production, and, when coupled with decreased survival of circulating red cells, results in severe anemia. As in other disorders characterized by ineffective erythropoiesis, β-thalassemia leads to inappropriately enhanced absorption of iron from the gut and progressive iron overload. As in most other types of severe anemia, erythropoietin production is markedly increased, resulting in the expansion of erythroid precursors not only in the bone marrow but also in extramedullary sites such as the liver and spleen. The enhanced proliferation of erythroid cells inside marrow cavities can lead to bony abnormalities described in the following section.

CLINICAL MANIFESTATIONS

β-Thalassemia Heterozygotes

Individuals with β-thalassemia trait are asymptomatic and have a normal life expectancy. Although most have normal hemoglobin levels, many are slightly anemic. All individuals with β-thalassemia trait have microcytosis with MCVs in the range of 75 to 80 fL. Some have elevated non-conjugated bilirubin levels, reflecting enhanced erythroid cell destruction due to a modest degree of ineffective erythropoiesis. In a small minority of heterozygotes, the spleen is slightly enlarged. In addition to microcytosis, the blood smear reveals red cell stippling and target cells (Fig. 8-7). The diagnosis of β-thalassemia trait is usually made by first ruling out iron deficiency and then demonstrating an elevation of Hb A₂ levels by electrophoresis. A small fraction of individuals with the phenotype of β-thalassemia trait have an allele in which the δ and β genes are both deleted. They have low or normal Hb A₂ levels and a modest elevation in Hb F. Individuals with β-thalassemia trait require no treatment and need to be reassured that this condition is benign and does not pose a health problem. Potential parents should be counseled about the risk of having a severely affected child if both partners have β-thalassemia trait.

β-Thalassemia Major (Cooley Anemia)

Individuals at the severe end of the clinical spectrum (often designated as having Cooley anemia) have marked anemia and, unless given meticulous medical care, usually do not survive beyond childhood. As depicted in Figure 8-6, the marked, sustained erythropoietin-mediated stimulus to red cell production leads to extramedullary erythropoiesis and enlargement of the liver and spleen. Iron overload develops because of enhanced absorption of iron from the gastrointestinal tract coupled with accumulation of iron following blood transfusion. Fair-skinned patients often have a light bronze appearance, owing to a combination of pallor, icterus, and enhanced skin pigmentation. The extension of massive erythroid hyperplasia into bone marrow cavities in the skull causes deformities such as expansion of the frontal bones and/or maxillary bones ("chipmunk face"). Enlargement of the mandible can result in malocclusion of the jaw. Because of multiple-organ and multiple-system involvement, symptoms are varied and complex but are generally centered on the anemia and cardiac failure. In particular, sustained high cardiac output can lead to heart failure unless patients are adequately transfused. If iron overload is not treated, patients develop life-threatening cardiomyopathy, along with hepatic fibrosis and endocrine failure, particularly of the pituitary gland and gonads. The expansion of erythroid marrow into the peripheral skeleton leads to osteopenia and occasionally pathologic fractures of long bones (Fig. 8-8).

The diagnosis of β-thalassemia major is usually straightforward. Severe transfusion-dependent anemia begins at approximately age 6 months with the switch from γ-globin to β-globin expression. The red cells (shown in Fig. 8-9) are extremely microcytic (MCV about 55-70 fL) and show huge variations in size and shape. Normoblasts are present in the peripheral blood, and their numbers are markedly increased in patients who have had a splenectomy (Fig. 8-9 B). In patients with β⁰/β⁰ thalassemia, nearly all of the hemoglobin is Hb F. Patients who are β⁺/β⁰ or β⁺/β⁺ have a variable amount of Hb A that accompanies the Hb F. Because of both enhanced absorption from the gut and blood transfusions, serum iron, transferrin saturation, and ferritin are all elevated.

The antenatal diagnosis of homozygous or compound heterozygous β-thalassemia can be established from analysis of DNA obtained by chorionic villous biopsy. The marked heterogeneity of β-thalassemia genotypes makes antenatal diagnosis technically challenging. Moreover, even if a diagnosis is established, the unpredictable degree of clinical severity, religious and cultural considerations, and the development of new and better therapies may dissuade parents from interrupting the pregnancy. Despite these caveats, in selected areas and with well-organized surveillance, antenatal diagnosis has been remarkably effective in drastically lowering the number of babies born with severe β-thalassemia.

Patients with β-thalassemia major require meticulous multi-disciplinary care. The mainstay of treatment is red cell transfusion sufficient to maintain hemoglobin levels above 10 g/dL. Many patients benefit substantially from splenectomy, which improves survival of endogenous red cells and therefore reduces the transfusion requirement. An adequate transfusion program will prevent the development of skeletal deformities, enhance growth and development, and obviate high output cardiac failure. However, transfusions pose a risk of infections such as HIV and hepatitis and development of antibodies (immunization) that may complicate matching of blood. Of even greater concern, transfusions inexorably increase the rate at which iron overload develops. Therefore all patients must be treated with iron chelators sufficient to put the patients into negative iron balance. Recent development of effective oral chelating agents may alleviate the need for daily subcutaneous administration.

Although chronic transfusion and chelation therapy are remarkably effective in preventing the morbid complications mentioned earlier, it is not only burdensome for the patient and his/her care providers but also imposes a huge economic burden. Stem cell transplantation, covered in detail in Chapter 26, offers a compelling alternative. Figure 8-10 shows a survival plot of a large number of patients with β-thalassemia major from Italy. About three-fourths of the patients were successfully transplanted without serious consequences over a period of up to 20 years. These patients can be considered cured. Moreover, despite the large initial financial outlay, stem cell transplantation is highly cost-effective over time. The chief limitation is that only about 25% of thalassemia patients have compatible stem cell donors. Alternative approaches to reverse the pathophysiology of β-thalassemia major include the development of pharmacologic agents that induce γ-globin expression and, further down the road, effective gene therapy.

β-Thalassemia Intermedia

Some individuals who inherit a β-thalassemia gene from each parent have clinical manifestations and complications less severe than those seen in β-thalassemia major and are designated as having β-thalassemia intermedia. In some cases, the milder clinical phenotype can be explained by: β⁺-thalassemia genes that allow expression of Hb A; high expression of γ-globin genes; or co-inheritance of β-thalassemia, which ameliorates globin chain imbalance. Patients with β-thalassemia intermedia by definition are not transfusion dependent. However, they are generally somewhat symptomatic from anemia and nearly always develop clinically significant iron overload as well as bone changes. For unclear reasons, many of these patients develop progressive pulmonary hypertension leading to cor pulmonale.

Interaction of β-Thalassemia With Common Hemoglobin Variants

The two most common structural hemoglobin variants are Hb E (α₂β₂^26glu→lys) and Hb S (α₂β₂^6glu→val). Compound heterozygous individuals who inherit a β-thalassemia gene from one parent and one of these structural variants from the other are commonly encountered and generally have a severe clinical phenotype. The β^E mutation is a single base substitution at the boundary between exon 1 and intron 1, leading to impaired splicing and therefore lowered β-globin expression. Accordingly, Hb E produces a phenotype resembling a very mild form of β⁺-thalassemia. Individuals who are either heterozygous or homozygous for Hb E have microcytosis but no significant clinical manifestations. In contrast, individuals who are β^E/β-thalassemia compound heterozygotes often have a severe phenotype indistinguishable from that of β-thalassemia major. Because the gene frequency of β^E is very high in populous southeastern Asia, β^E/β-thalassemia is commonly encountered and is a major cause of morbidity and early mortality.

The gene frequency of β^S is comparably high in central Africa, Arabia, and India. Accordingly, β^S/β-thalassemia compound heterozygotes are commonly encountered in these regions as well as in areas to which central Africans have migrated, such as Italy and Greece. Because the β-thalassemia allele expresses either no or very little β^A, the major hemoglobin component in β^S/β-thalassemia red cells is Hb S, and therefore the potential for sickling in these cells is greatly enhanced. As explained in Chapter 9, patients with the genotype β^S/ β⁰ have clinical manifestations as severe as those of sickle cell (β^S/ β^S) patients, whereas those with the genotype β^S/β⁺ have significantly milder manifestations.

More than 10 million individuals around the world are heterozygote carriers of an α-thalassemia gene, a prevalence somewhat less than that of β-thalassemia. Affected individuals are predominantly from tropical areas in Asia and Africa. As in β-thalassemia, there is strong epidemiologic evidence that inheritance of a single β-thalassemia gene confers protection against falciparum malaria. Gene deletion accounts for the vast majority of β-thalassemia. As depicted in Figure 8-11, deletions have arisen because of unequal meiotic crossover between adjacent α-globin genes. Because individuals normally inherit two α-globin genes, one from each parent, there are four types of α-thalassemia, depending on how many genes have been deleted (Table 8-1). The most common abnormal haplotype is –α, in which there is only one functioning α gene. About 30% of black Africans are heterozygous for this genotype. Thus, they have a deletion of one of four α-globin genes. In the southern Mediterranean and in Southeast Asia, –α is also very common. The – – haplotype involves deletion of both of the tandem α-globin genes. This genotype is common in southeastern Asia but rare elsewhere (Fig. 8-11).

The various genotypes in α-thalassemia (–α/αα, –α/–α, – –/αα, – –/–α, and – –/– –) correlate with the clinical phenotypes in accord with the number of α-globin genes deleted. Individuals with a single α-globin gene deletion (–α/αα) are called silent carriers. They have an entirely normal clinical and hematologic phenotype except for slight microcytosis. As shown in Figure 8-4, measurement of globin synthesis in reticulocytes shows a modest increase in the β/α ratio compared with normals. Individuals with two-deletion α-thalassemia may either be homozygous for the –α allele or heterozygous for the – – allele. The two types of two-deletion α-thalassemias have the same clinical phenotype: they are entirely asymptomatic and have normal or near-normal hemoglobin levels with significant microcytosis, occasional target cells, and an increased β/α synthesis ratio (Fig. 8-4). Two-deletion α-thalassemia should be suspected in African and Asian individuals with no or minimal anemia and moderate microcytosis, once iron deficiency and β-thalassemia trait have been ruled out by appropriate laboratory tests.

As shown in Table 8-1, the more serious forms of α-thalassemia require the inheritance of a – – allele with a –α allele (Hb H disease, – –/–α) or with another – – allele (hydrops fetalis, – –/– –) and are therefore seen almost exclusively in Southeast Asians. Although Hb H disease and hydrops fetalis are much less commonly encountered than the mild forms of α-thalassemia, awareness of these conditions in the United States has been heightened by the influx of large numbers of refugees following the Vietnam War. Individuals with Hb H disease have prominent microcytosis and target cells, shown in Figure 8-12, along with a marked increase in β/α synthesis (Fig. 8-4). Because of the large decrement in α-globin production, excess γ-globin accumulates in red cells of the fetus, newborn, and infant and assembles to form an abnormal hemoglobin tetramer known as Hb Bart (γ₄). Following the switch in globin expression in later infancy (Fig. 8-2), the red cells contain an excess of β-globin, which self-associates to form another abnormal hemoglobin, Hb H (β₄). Hb H is relatively insoluble and forms intracellular precipitates called Heinz bodies, which impair red cell deformability and cause premature destruction by macrophages. Thus, individuals with Hb H disease have moderate (and occasionally severe) hemolytic anemia. The treatment for this condition is primarily supportive. If significant iron overload occurs, it should be treated with iron chelation. Splenectomy is of doubtful efficacy.

The inheritance of a – – allele from each parent results in the total absence of α-globin and thus a complete inability to produce Hb F and Hb A. As a result, during gestation nearly all of the hemoglobin is Hb Bart (γ₄). In order for hemoglobin to transport oxygen efficiently, it must be a heterotetramer composed of two pairs of structurally distinct subunits (e.g., α + γ or α + β). In contrast, homotetramers such as Hb Bart have a high affinity for oxygen and lack subunit cooperativity. Even though the blood of a fetus with – –/– – thalassemia is fully oxygenated, it does not release the oxygen to tissues, and therefore the baby suffers from severe tissue hypoxia. At term it is markedly edematous (Fig. 8-13) and moribund, and thus is either stillborn or dies a day or so after birth. The blood morphology reveals dramatic microcytosis, abnormalities in red cell shape, and a profusion of immature nucleated erythroid cells, as shown in Figure 8-14. The possibility of having a – –/– – fetus should be anticipated in Asian families where one or both parents have microcytosis and especially when there has been a previous baby with hydrops fetalis. Reliable antenatal diagnosis can be done by DNA analysis on a chorionic villus biopsy. If – –/– – α-thalassemia is established early in pregnancy, most parents will elect to terminate the pregnancy. If the diagnosis is not made until the second trimester or early in the third trimester, the fetus can be salvaged by intrauterine blood transfusions. Following birth, stem cell transplantation (Chapter 25), if successful, can be curative, but a compatible donor is found only in about 25% of cases.