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.
Assembly of hemoglobin subunits and hemoglobin tetramer in β-thalassemia. ROS = reactive oxygen species; PPT = hemoglobin precipitate. (Modified with permission from Yu Y, Kong Y, Dore LC et al, An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis, J Clin Invest 2007, 117: 1856-1865.)
Flow diagram summarizing the cellular pathogenesis of β-thalassemia.
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.
Blood smear from an individual with β-thalassemia minor.
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).
Radiographs of the distal long bones in the arm and leg of a child with β-thalassemia major.
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.
Blood smear from a patient with β-thalassemia major before (A) and after (B) splenectomy.
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.
Disease-free survival plot of 866 patients with β-thalassemia major following stem cell transplantation from HLA-identical donors.
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.
β-Thalassemia Rx: Splenectomy
Stem cell transplant
Induction of Hb F
Interaction of β-Thalassemia With Common Hemoglobin Variants
The two most common structural hemoglobin variants are Hb E (α2β226glu→lys) and Hb S (α2β26glu→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 βE 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 βE/β-thalassemia compound heterozygotes often have a severe phenotype indistinguishable from that of β-thalassemia major. Because the gene frequency of βE is very high in populous southeastern Asia, βE/β-thalassemia is commonly encountered and is a major cause of morbidity and early mortality.
The gene frequency of βS is comparably high in central Africa, Arabia, and India. Accordingly, βS/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 βA, the major hemoglobin component in βS/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 βS/ β0 have clinical manifestations as severe as those of sickle cell (βS/ βS) patients, whereas those with the genotype βS/β+ have significantly milder manifestations.