Background

Thalassemia was first recognized by Cooley and Lee in 1925¹⁸ as a form of severe anemia associated with splenomegaly and bone changes in children. The term thalassemia is derived from the Greek φαλασσα (the sea) since many of the early cases came from the Mediterranean region. However, it is now clear that the disorder is not limited to the countries around the Mediterranean but occurs throughout the world, being also prevalent in the tropical and subtropical regions including the Middle East, parts of Africa, the Indian subcontinent and Southeast Asia. It appears that heterozygotes for thalassemia are protected from the severe effects of falciparum malaria and natural selection has increased and maintained their gene frequencies in these malarious regions.

The thalassemias are classified into α, β, δβ, γδβ, δ, γ and εγδβ thalassemias according to the type of globin chain(s) that is reduced. The two major categories are the α and β thalassemias while the rare forms include the δβ, γδβ and εγδβ thalassemias. Hereditary persistence of fetal hemoglobin (HPFH) refers to the group of disorders in which the switch from fetal to adult hemoglobin production is altered and high levels of fetal hemoglobin are synthesized in individuals. Because of their concomitant increase in HbF levels, the δβ and γδβ thalassemias are often considered with the HPFH syndromes. In many populations the α and β thalassemias coexist with a variety of different structural hemoglobin variants. In these populations it is quite common to inherit a combination of α and/or β thalassemia and/or structural hemoglobin variant genes; these complex interactions give rise to an extremely wide spectrum of clinical phenotypes which together constitute the thalassemia syndromes.¹⁰

Most thalassemias are inherited in a Mendelian recessive fashion. Heterozygotes are normally symptomless, although they can often be recognized by simple hematological analysis. Severely affected individuals have inherited two copies of mutant hemoglobin gene, homozygotes for α or β thalassemia, or compound heterozygotes for different molecular forms of α or β thalassemia and a hemoglobin variant. It has been estimated that about 300 000 individuals severely affected with thalassemia are born each year, posing a heavy burden on the health services.¹⁰ Due to recent population movements, these hemoglobin disorders have become an important part of clinical practice in all countries, including the UK.¹⁹

Genetic basis of disease

Molecular analysis of the β thalassemia genes has demonstrated a striking heterogeneity. Although almost 300 β thalassemia alleles (including deletions) have been characterized, population studies indicate that probably only 20 β thalassemia alleles account for >80% of the β thalassemia mutations in the whole world.^1,10 This is because in each of the high-frequency areas, only a few (4–6) mutations are common, reflecting the local selection from malaria, with a varying number of rare ones. Each of these populations has its own unique group of mutations.

Unlike α thalassemia, in which deletions in the α-globin gene cluster account for most of the mutations, the molecular defects causing β thalassemia are usually point mutations involving one (or a limited number of nucleotides) within the β gene or its immediate flanking regions.^1,10,20

These point mutations involve the critical sequences that interfere with gene function at either the transcriptional or the post-transcriptional level, including translation (Fig. 9.2). Approximately half of these mutations completely inactivate the β gene with no β-globin production causing β° thalassemia. Other mutations allow the production of some β-globin, and are classified as β⁺ or β⁺⁺ thalassemia, depending on the degree of quantitative reduction in the output of the β chains. The β-globin chains that are synthesized are usually structurally normal. Mutations affecting the conserved sequences in the 5′ promoter, i.e. TATA box, proximal CACCC and distal CACCC box, typically cause a 70–80% reduction in promoter activity and are often very mild. Mutations affecting the polyadenylation signal (AATAAA) at the 3′ end, also generally result in a mild β⁺ thalassemia phenotype.

Fig. 9.2 Point mutations causing β thalassemia. The β globin gene is represented by 3 exons (gray) interrupted by 2 introns with the 5′ and 3′ untranslated regions (UTRs, striped boxes). The vertical lines within the open rectangular boxes represent the sites of the different mutations which can be found in the UTRs, exons and introns, and causing typical recessively inherited β thalassemia. Mutations in the hatched rectangular boxes are dominantly inherited. They include mutations that lead to premature termination due to frameshifts or nonsense codons, insertions or deletions of intact codons and amino acid substitutions.

A few β thalassemia mutations are ‘silent’; carriers do not have any evident hematological phenotypes with near normal red cell indices and HbA₂ levels, the only abnormality being an imbalanced globin chain synthesis. These β thalassemia mutations have usually been ascertained by finding individuals with intermediate forms of β thalassemia resulting from compound heterozygosity for one typical β thalassemia mutation in combination with a very mild β⁺ thalassemia allele. In this case, one parent has typical β° thalassemia trait and the other, apparently normal. Overall, the ‘silent’ β° thalassemia alleles are uncommon except for the –101 C→T mutation which has been observed fairly frequently in the Mediterranean region where it interacts with a variety of more severe β thalassemia mutations to produce milder forms of β thalassemia.²¹

The β° thalassemia alleles are mostly caused by premature termination of translation, either by single base substitution to a nonsense codon, or through a frameshift mutation. Studies show that the different in-phase termination mutants exhibit a ‘positional’ effect and are subjected to a surveillance mechanism (nonsense mediated RNA decay or NMD) to prevent the accumulation of mutant mRNAs coding for truncated peptides.^22,23 Frameshifts and nonsense mutations that result in premature termination early in the sequence (in exon 1 and 2) are associated with minimal amounts of mutant β-mRNA.²⁴ In such cases, no β chain is produced from the mutant allele, resulting in a phenotype of typical heterozygous β thalassemia in the carrier. However, some mutations that produce in-phase terminations later in the β sequence, in exon 3 and the 3′ half of exon 2²⁵ escape NMD, and are associated with substantial amounts of abnormal β-mRNA from the mutant allele. The mutant β-mRNA leads to a synthesis of abnormal β chain variants that are often highly unstable and non-functional, and are not able to form viable tetramers, thus effectively causing a functional deficiency of β-globin chain.¹ Not only are these abnormal β-globin chain variants non-functional, but they prove to be an additional nuisance as they precipitate in the erythroid precursors, overloading the proteolytic mechanism and accentuating the ineffective erythropoiesis. Carriers for such β thalassemia alleles have fairly severe anemias, hence the term ‘dominantly inherited β thalassemia’. For a detailed review of the dominantly inherited β thalassemias, see Thein & Wood 2009¹ and Thein 1999.²⁶

β thalassemia is rarely caused by deletions. Of these, only the 619 bp deletion at the 3′ end of the β gene is common, but even that is restricted to the Sind populations of India and Pakistan where it constitutes ~30% of the β thalassemia alleles.^1,27 The other deletions, although extremely rare, are of particular clinical interest because they are associated with an unusually high level of HbA₂ in heterozygotes. The mechanism underlying the markedly elevated levels of HbA₂ and the variable increases in HbF in heterozygotes for these deletions is postulated to be related to the removal of the 5′ promoter region of the β-globin gene which removes competition for the upstream β-LCR and limiting transcription factors, resulting in an increased interaction of the LCR with the γ and δ genes in cis, thus enhancing their expression (Fig. 9.3). This mechanism may also explain the unusually high HbA₂ levels associated with the promoter mutations at positions –88 and –29.

Fig. 9.3 Deletions causing β thalassemia. The deletions that remove the promoter region are associated with unusually high HbA₂ levels and variably increased HbF levels in the heterozygous state.

Unusual causes of β thalassemia, although rare, illustrate the numerous molecular mechanisms of down-regulating the β-globin gene.¹ They include transposable elements which may disrupt and inactivate human genes. The insertion of such an element, a retrotransposon of the LI family into intron 2 of the β-globin gene, has been reported to cause β⁺ thalassemia.²⁸ Mutations in other genes distinct from the β-globin complex (trans-acting mutations) can down-regulate β-globin expression. Such trans-acting mutations have been described affecting the XPD protein that is part of the general transcription factor TF11H,²⁹ and the erythroid-specific GATA-1 protein.³⁰ Somatic deletions of the β-globin gene have been reported as the contributory cause for the unusually severe anemia in three unrelated families of French and Italian origin^31,32 (see intermediate forms of β thalassemia). Unipaternal isodisomy of chromosome 11p15.5 that encompassed the β-globin gene cluster contributed to thalassemia major in a Chinese patient.³³

Pathophysiology

The molecular defects in β thalassemias result in absent or reduced β chain production while α chain synthesis proceeds at a normal rate. This imbalance in globin synthesis in β thalassemia gives rise to excess α chains which are extremely unstable and precipitate in the red cell precursors forming inclusion bodies (Fig. 9.4). These inclusions interfere with the red cell maturation and are responsible for the intramedullary destruction of the erythroid precursors and hence the ineffective erythropoiesis that characterizes all β thalassemias. The anemia of β thalassemia results from a combination of underproduction of hemoglobin and ineffective erythropoiesis and, ultimately, correlates well with the degree of α- to non α-globin chain imbalance and the amount of free α chains. The complications of splenomegaly, bone disease, endocrine and cardiac damage are related to the severity of anemia and the hypercatabolic state, and the subsequent degree of iron loading resulting from the increased gastrointestinal iron absorption and from repeated blood transfusion.

Fig. 9.4 Diagrammatic representation of the pathophysiology of β thalassemia.

Genotype-phenotype correlation

Typically, β thalassemia is inherited as haploinsufficient Mendelian recessives. The most severe end of the clinical spectrum, β° thalassemia, is characterized by the complete absence of HbA (α₂β₂) and results from the inheritance of two β° thalassemia alleles (homozygous or compound heterozygous states). This normally results in the transfusion-dependent state of β thalassemia major and, at worst, the patients present within 6 months of life with profound anemia and, if not treated with regular blood transfusions, die within their first 2 years. Individuals who have inherited a single β thalassemia allele, whether β° or β⁺, have thalassemia trait. They are clinically asymptomatic but may have a mild anemia with characteristic hypochromic microcytic red blood cells, elevated levels of HbA₂ (α₂δ₂) and variable increases of HbF (α₂γ₂). Inheritance of two β thalassemia alleles, however, does not always lead to thalassemia major; many patients with two β thalassemia alleles have a milder disease, ranging from a condition that is slightly less severe than transfusion-dependence to one that is asymptomatic and often mistaken for thalassemia trait. The diverse collection of phenotypes between the two extremes of thalassemia major and trait constitute the clinical syndrome of thalassemia intermedia.

The heterozygous states for β thalassemia also show a tremendous phenotypic diversity, comparable to that for the inheritance of two β thalassemia alleles. In some cases, the β thalassemia allele is so mild that it is phenotypically ‘silent,’ with no anemia or hematological abnormalities. In others, the heterozygous state causes a phenotype almost as severe as the major forms, that is, the β thalassemia allele is dominantly inherited.

Unraveling the molecular basis of the clinical diversity in the intermediate state has provided tremendous insights on genotype-phenotype correlation and clues for the development of molecular therapy for the β thalassemias.

The main genetic interactions that result in a phenotype of thalassemia intermedia are summarized in Table 9.1. Thalassemia intermedia can result from the inheritance of one or two β thalassemia alleles. In 60–90% of the cases^34–36 the patients have inherited two β thalassemia alleles and the reduced disease severity can be explained by the inheritance of the milder forms (β⁺⁺ and ‘silent’ β thalassemia alleles) that allow the production of a significant proportion of β-globin chains. Co-inheritance of a single α-globin gene deletion (αα/α−) has very little effect on β° thalassemia, but individuals with two α-globin gene deletions (α−/α− or αα/−−) and β⁺ thalassemia have a mild disease requiring intermittent transfusions. The role of increased HbF response as an ameliorating factor becomes evident in the group of thalassemia intermedia patients who are mildly affected despite having minimal amounts or no HbA (α2β2), and without α thalassemia. Three major QTLs – Xmn1-Gγ, HBS1L-MYB on chromosome 6q, and BCL11A on chromosome 2p – recently identified in genome-wide association studies (GWAS) have been shown to impact HbF levels, not only in healthy individuals, but also patients with β thalassemia and sickle cell disease (SCD).^13,37,38 In Sardinia, BCL11A and HBS1L-MYB with α thalassemia accounts for 75% of variable phenotypic severity of β thalassemia,³⁹ and in Thailand, the three HbF QTLs are associated with both disease severity and HbF levels in HbE/β thalassemia.⁴⁰

Table 9.1 Thalassemia intermedia: the common genetic interactions that underlie the phenotype of thalassemia intermedia

Other HbF determinants within the cluster are related to the mutation itself. Small deletions or mutations that involve the promoter sequence of the β-globin gene are associated with variable increases in HbF and unusually high HbA₂ levels, and reflect the competition between the γ and β globin gene promoters for interaction with the upstream β-LCR. Hence, although such deletions cause a complete absence of β-globin product, the severity of the phenotype is offset by the concomitant increase in hemoglobin F.

Inheritance of single copies of β thalassemia gene can also lead to thalassemia intermedia. In the majority of cases this is caused by the co-inheritance of extra α-globin genes, the severity of outcome depends on the total number of α-globin genes inherited (from one or two copies of triplicated (/ααα), or quadruplicated (/αααα) α-globin complexes), and the type of β thalassemia mutation (β° or β⁺).^41,42 More recently, another mechanism of inheriting extra α-globin genes has been described which involved segmental duplication of the whole α-globin gene cluster.^43,44 The additional α-globin genes have no phenotype in normal people but the small excess of α chains in heterozygous β thalassemia appears to tip the balance, crossing the critical threshold of α-globin excess with phenotypic consequences.

In a number of cases, the unusually severe heterozygous state is associated with a normal α-globin genotype. In such cases, the β thalassemia mutation itself leads to the synthesis of highly unstable, structurally abnormal β-globin chain variants.¹⁷ The hyperunstable β chain variants are rapidly destroyed in the erythroid precursors, giving rise to a functional deficiency of β chains and simulating a phenotype of β thalassemia. The non-functional β chain variants together with the unmatched α-globin chains aggravate the ineffective erythropoiesis causing a disease phenotype even when present in a single copy. Hence, the term ‘dominantly inherited β thalassemia’.

Finally, although rare, somatic mosaicisms of the β-globin gene in β thalassemia heterozygotes have been reported causing thalassemia intermedia. The individuals from three different families had moderately severe anemia despite being constitutionally heterozygous for a common β thalassemia mutation; they also had a normal α (αα/αα) genotype.^31,32 Subsequent investigations revealed that these individuals had a somatic deletion including the β-globin complex, on the other chromosome 11p15, in a subpopulation (~80%) of erythroid cells giving rise to a somatic mosaic: about 20% of the erythroid cells were heterozygous with one normal copy of β-globin gene and the rest hemizigous (i.e. without any normal β-globin gene). The sum total of the β-globin product is thus about 80% less than the asymptomatic β thalassemia trait. These unusual cases once again illustrate that the severity of anemia in β thalassemia reflects the quantitative deficiency of β-globin production.

Given the differences in the spectrum of β thalassemia mutations and differences in the frequency of the different α thalassemia variants, the relative importance of these genetic factors would vary accordingly in different population groups. It is also important to note that the genotypic factors are not mutually exclusive.

Tertiary modifiers affect complications of the disease; the severity of osteopenia and osteoporosis, iron loading and jaundice may be affected by polymorphisms of genes involved in the metabolic pathways concerned with these complications.⁴⁵ Bone mass is a quantitative trait under strong genetic control involving multiple quantitative trait loci (QTLs) and the QTLs implicated include estrogen receptor gene, vitamin D receptor (VDR), collagen type a1 genes and transforming growth factor β1 (TGFβ1).⁴⁶ The elevations of bilirubin and incidence of gallstones in thalassemia, as in other hemolytic anemias, are influenced by a polymorphic variant (seven (TA) repeats) in the promoter of the uridine diphosphate-glucuronosyltransferase 1A (UGT1A1) gene, also referred to as Gilbert’s syndrome.^47,48 Iron loading in β thalassemia is also variable and results not just from blood transfusion but also from increased iron absorption. Variants in the HFE gene have a modulating effect on iron absorption,⁴⁹ and as other genes in iron homeostasis become uncovered, it is likely that there will be genetic variants in these loci that influence the different degrees of iron loading in β thalassemia.⁵⁰ For instance, recent genome wide association studies have identified variants in HFE, and the TMPRSS6 (transmembrane protease serine 6 gene that regulates hepcidin expression) associated with iron status, erythrocyte volume and concentration.^51–53

Diagnosis

In β thalassemia major, untransfused hemoglobin levels are usually less than 5 g/dl but can be as low as 2–3 g/dl. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are low, with a very wide red cell distribution width (RDW), marked anisopoikilocytosis, target cell formation, and basophilic stippling. Poorly hemoglobinized nucleated red cells are frequently found in the peripheral blood, and may reach very high levels after splenectomy. The reticulocyte count is elevated but less than expected for the degree of anemia in keeping with the ineffective erythropoiesis. A bone marrow aspirate is not essential to make the diagnosis, but if performed shows marked erythroid hyperplasia characterized by poorly hemoglobinized normoblasts and dyserythropoiesis. Supravital staining (e.g. methyl violet) shows ragged inclusions in many of the erythroid precursor cells; similar inclusions are found in the peripheral red blood cells after splenectomy. Immunoelectron microscopy confirms that these inclusions consist of precipitated α-globin chains (Figs 9.5 and 9.6). Increased iron deposition is also seen in the bone marrow; the majority of the iron granules are randomly distributed. Biochemical evidence of hemolysis such as elevated bilirubin, aspartate transaminase (AST) and lactate dehydrogenase (LDH) with a normal alanine transaminase (ALT) and progressive iron loading is observed. Other biochemical changes may include evidence of diabetes and endocrine dysfunction such as parathyroid or thyroid insufficiency.

Fig. 9.5 Electron micrograph of a late erythroblast from a homozygote for β thalassemia. The cytoplasm contains multiple small rounded masses of electron-dense material, some of which have fused together to form larger masses. The masses probably consist of precipitated α chains. Uranyl acetate and lead citrate. × 11 625.

(Courtesy of Professor SN Wickramasinghe).

Fig. 9.6 Bone marrow macrophage from a case of homozygous β thalassemia. The macrophage contains a phagocytosed late erythroblast within which intracytoplasmic α chain precipitates can be recognized. Uranyl acetate and lead citrate. × 11 150.

(Courtesy of Professor SN Wickramasinghe).

Hemoglobin analysis is needed to confirm the diagnosis, typically using electrophoretic or chromatographic techniques. The findings on Hb electrophoresis vary with the β thalassemia genotype and are informative only in the previously untransfused patient. Absence of HbA in pretransfused samples confirms a diagnosis of β° thalassemia, and the hemoglobin consists of F and A₂ only. In β⁺ thalassemia (homozygous or compound heterozygotes), a variable amount of HbA is present. The HbF is usually elevated and varies from 10 to almost 100% of the total hemoglobin in the case of β° thalassemia. The HbA₂ level is of no diagnostic value. In vitro globin chain biosynthesis of peripheral blood reticulocytes or bone marrow shows globin chain imbalance with a marked excess of α over β and γ chain production. Obviously, in β° thalassemia there is a complete absence of β-globin chain synthesis.

The heterozygous state for β thalassemia (β° or β⁺) is remarkably uniform hematologically. Anemia, if present, is mild and the diagnosis is based on a low mean corpuscular volume (MCV) and mean cell hemoglobin (MCH) accompanied by an increased proportion of HbA₂, from 3.5 to 5.5%, with the exception of a subgroup that has a normal level of HbA₂. Globin chain biosynthesis shows α chains in excess of about twofold. The hematological and hemoglobin electrophoresis profiles are usually sufficient to make a diagnosis of β thalassemia, although DNA analysis, where available, is often used to confirm the diagnosis.

Normal HbA₂ heterozygous β thalassemia may be difficult to distinguish hematologically from heterozygous α thalassemia since both cases have low MCVs and low MCHs, and normal HbA₂ levels. The distinction is made by globin chain biosynthesis (where available) and DNA analysis; family studies are useful. Type 1 normal HbA₂ β thalassemia, otherwise known as ‘silent’ β thalassemia, has almost normal red cell indices; it results from very mild mutations which cause only a minimal deficit in β chain production. The other group of normal HbA₂ β thalassemia (type 2) results from the co-inheritance of δ thalassemia in cis or in trans to the β thalassemia gene; in the latter, family studies show independent segregation of the thalassemia indices and normal HbA₂ levels.

Clinical features and management

The clinical phenotypes of β thalassemia range from very severe (major) to a completely silent carrier state, with a huge range of intermediate phenotypes (thalassemia intermedia) between the two ends of the spectrum.¹⁰

Thalassemia major. In many developed countries, infants affected with severe forms of β thalassemia will be first identified by neonatal screening programs, before the development of any symptoms; antenatal screening of the parents and possibly prenatal diagnosis may have identified the infant to be at risk of severe β thalassemia before birth. The severely affected child is likely to require regular red blood cell transfusions. To meet the aims of correcting the anemia and suppressing the abnormal endogenous erythroid hyperplasia, the trough hemoglobin levels need to be above 9.5 g/dl. In practice, this means maintaining pretransfusion hemoglobin of 9–10 g/dl.

Inadequately transfused children develop the typical features of Cooley’s anemia. They show marked retardation of growth and development with progressive hepatosplenomegaly. A typical ‘thalassemic’ facies develops with frontal bossing, prominent cheek bones and protruding upper jaw due to erythroid hyperplasia in the skull and facial bones. Radiography of the skull shows the typical ‘hair-on-end’ appearance. The long bones and phalanges become rarefied from marrow expansion and show a lacy, trabecular pattern on radiography. These changes may be associated with repeated pathological fractures. Occasionally the expanding marrow extends from the rib or vertebrae and forms large paraspinal extramedullary masses. The massive marrow expansion causes a hypermetabolic state accompanied by intermittent fevers and weight loss. Gallstones and leg ulcers are common complications. Without any transfusion, death occurs within the first 2 years. ‘Palliative’ transfusion allows the child to live somewhat longer but the bony deformities remain unchanged and the child ultimately succumbs to an overwhelming infection. If these children survive to puberty, they develop complications of iron overload. Iron accumulation results from an increased rate of gastrointestinal absorption as well as that derived from the blood transfusions.

Adequately transfused children grow and develop normally until early puberty. Iron overload inevitably complicates regular blood transfusions, and progress of their disease then depends on whether they have received regular iron chelation. If not, they begin to show signs of progressive hepatic, cardiac and endocrine disturbances including diabetes, hypoparathyroidism and delayed or absent secondary sexual development. The endocrinopathies and cardiac disease are ascribed to the labile, more toxic forms of iron that appear in cells and plasma, referred to as non-transferrin bound iron (NTBI). Throughout their teenage life these children suffer from a variety of complications due to different endocrine deficiencies. Unless iron overload is controlled by regular chelation therapy, death results in the second or third decade, from acute or intractable congestive cardiac failure. Children who are adequately transfused and fully compliant with iron chelation therapy grow and develop normally, with few or no skeletal abnormalities, and achieve sexual maturity. Even within this group there is a high frequency of growth retardation and retarded sexual maturity, with variable complications relating to iron metabolism, bone disease, endocrine abnormalities and liver disease.⁵⁴

Iron chelation is usually started after 1 year of monthly blood transfusions.^55,56 Currently, three iron chelating agents are available for use: desferrioxamine (DFO), deferiprone and deferasirox. DFO has been in use since the 1970s and is known to be safe and effective; side-effects seem to occur when the drug is used in high doses or when iron stores are low. The main problem with DFO is that it has to be given parenterally, and due to its short half-life, the regimen involves continuous subcutaneous infusions given over 8 hours using a syringe driver or balloon pump. Deferiprone was the first oral iron chelator to be used; it was licensed in Europe in 1999. Arthropathy and agranulocytosis (which can be fatal) are potential serious side-effects, and patients taking deferiprone are recommended to have weekly blood counts. Deferiprone seems to be particularly effective in removing cardiac iron, a therapeutic effect ascribed to its low molecular weight. Deferiprone is increasingly used in combination with DFO.⁵⁷ Deferasirox is a once-daily oral iron chelator, approved in the USA and Europe since the mid-2000s.⁵⁸ Large clinical trials have demonstrated its efficacy in removing liver iron in thalassemia, with increasing evidence that it also removes cardiac iron. All patients on regular blood transfusion and iron chelation therapy should be monitored regularly with a yearly review for assessment of growth, including tests for endocrine and cardiac function, iron loading (serum ferritin, MRI for liver iron concentration) and adverse effects related to therapy.⁵⁶

Bone marrow transplantation (BMT) is considered the treatment of choice if there is a HLA-identical sibling. BMT should be considered only if it is clear that the child is transfusion-dependent and should be considered early as the success of BMT is reduced as the child gets older, with increasing iron overload and iron-related organ damage.⁵⁹

Thalassemia intermedia. Thalassemia intermedia (TI) includes patients with a wide range of clinical phenotypes. To a large extent, whether a patient is classified as thalassemia intermedia or major depends on a doctor deciding if the patients would benefit from regular blood transfusions; this decision is based not only on clinical factors (such as failure to grow and frequent infections) but also on non-clinical factors such as experience of the doctor, availability of blood and wishes of the patient.

The clinical sequelae of TI results from the ineffective erythropoiesis, chronic anemia and iron overload. The severity may change with increasing age due to iron loading from increased intestinal absorption and decreased tolerance of anemia due to reducing cardiovascular fitness. A wide range of other problems is particularly associated with TI, including pulmonary hypertension, hypercoagulability, pseudoxanthoma elasticum and other connective tissue disorders, hypersplenism, leg ulceration, folate deficiency, extramedullary hemopoietic tumor masses in the chest and skull, gallstones and a marked proneness to infection. There are currently no clear guidelines for managing patients with TI. Because of the extreme variability of these disorders, management is highly dependent on the course that is likely to evolve in an individual patient; all patients should be followed from early childhood and carefully monitored in terms of growth and iron loading.

Intermittent blood transfusions in TI are often necessary due to falls in hemoglobin caused by fever, infection and specifically human parvovirus B19 infection. It can be difficult to decide who would benefit from a short period of regular blood transfusions and when to start them. In countries with a ready supply of safe blood there is an increasing tendency to start regular transfusions, even in children maintaining hemoglobins greater than 8 g/dl, to avoid the emerging complications of skeletal deformities, pulmonary hypertension and osteopenia. There is also some evidence that this improves the quality of life, particularly with emerging options for oral iron chelation. This is not possible in much of the world and management consists of reserving transfusion for severe symptomatic anemia. The initiation of iron chelation depends on the degree of iron overload (as indicated by liver iron concentration), but as with other aspects of the management of TI, there are no clear guidelines.⁶⁰

Pharmacological treatment to increase HbF and total hemoglobin levels is potentially applicable to TI, in that relatively small increases in hemoglobin levels with a corresponding reduction in ineffective erythropoiesis could help a patient thrive who would otherwise require regular transfusions.¹ Hydroxyurea is the most widely used drug in this context, with encouraging results in some patients. Butyrate and other short-chain fatty acid derivatives also promote HbF synthesis, and have been used with limited clinical success in TI. A number of newer drugs are being developed which may boost HbF to a greater extent, most notably the new generation of short-chain fatty acid derivatives (SCFADs) and immunomodulatory drugs such as pomalidomide and lenalidomide.

Thalassemia minor. Individuals with β thalassemia minor (i.e. carriers) are typically asymptomatic. Splenomegaly is rare.

Disease Prevention. The thalassemias are a major health problem in many populations. Apart from bone marrow transplantation, there is no definitive treatment; hence major efforts are concentrated on prevention of the disease.

Preventive programs in most countries now combine education, pre-conceptual, antenatal and neonatal screening, heterozygote detection and genetic counseling for a comprehensive approach in the public health management of the disease.⁶¹ Many screening programs are centered at antenatal clinics and concentrate on identifying women who are thalassemia carriers in the first trimester of pregnancy. This is done by varying combinations of blood tests and identifying women at high risk of carrying thalassemia based on their ethnic origin; this latter approach is particularly effective in areas with a low prevalence of thalassemia in the native population, such as northern Europe. If a woman is found to be a carrier, screening is then offered to her partner, and if both are carriers, they are counseled about the risk of the fetus inheriting a severe form of thalassemia and offered prenatal diagnosis, usually from 11–12 weeks’ gestation by chorionic villus sampling or amniocentesis. Parents can then make an informed choice to terminate the pregnancy if the fetus is affected. However, chorionic villus sampling and amniocentesis are invasive with an increased risk of miscarriage of about 1%. This has led to research to develop non-invasive methods of prenatal diagnosis based on maternal blood sampling. Maternal blood contains small numbers of fetal cells and also cell-free fetal DNA, both of which could potentially be used to diagnose fetal thalassemia.

Some couples at risk of having an affected child find prenatal diagnosis and selective termination unacceptable. Pre-implantation genetic diagnosis (PGD) involves the use of in vitro fertilization techniques to generate 5–15 embryos; at the eight-cell stage, one embryonic cell can be removed and tested for thalassemia alleles; it is then possible to implant only embryos without thalassemia.⁶² PGD, however, is currently a difficult, stressful and expensive procedure, with only 10–20% of couples taking home a baby.

Newborn screening detects the majority of babies with severe thalassemia, either using cord blood or, more commonly, from the neonatal blood spot; this is taken onto a piece of blotting paper and also screened for other conditions such as phenylketonuria. If hemoglobin analysis shows only HbF, with no HbA or other hemoglobin variants, it is likely that the baby has inherited a severe form of β thalassemia and may be transfusion-dependent; less severe possibilities include thalassemia intermedia and homozygosity for hereditary persistence of fetal hemoglobin. Babies identified in this way can then be followed up closely rather than waiting until they present following a period of prolonged illness. Parents can also be tested and given advice concerning the risk to future pregnancies.

HbE/β thalassemia

This is the commonest severe form of thalassemia in Southeast Asia and parts of the Indian subcontinent. Recent demographic changes, however, have resulted in HbE/β thalassemia becoming a health problem in other parts of the world, such as North America.⁶³

The β^E allele is mildly thalassemic as the mutation at β codon 26 (GAC→AAG, Glu→Lys) that gives rise to HbE, also activates a cryptic splice site, and when inherited together with β° thalassemia, results in a marked deficiency of β chain production. The clinical and hematological changes of HbE /β thalassemia are variable, ranging from severe anemia and transfusion dependency to thalassemia intermedia.^10,63 There is nearly always anemia (hemoglobin values range from 4–9 g/dl) and splenomegaly.

While a large part of this phenotypic variability can be explained by the severity of the β thalassemia alleles, this cannot be the only answer since an equally broad range of clinical phenotypes has been encountered in HbE/β° thalassemic individuals who all carry null β thalassemia mutations.^1,64 It can be difficult to differentiate homozygous HbE (HbE/E) from HbE/β° thalassemia on Hb electrophoresis alone since only HbE and F are observed in both cases. Genetic studies would be definitive since both parents would be HbE carriers in HbE/E, but HbE trait in one parent and β thalassemia trait in the other, in HbE/β thalassemia. Clinically, homozygotes for HbE have mild anemia and are asymptomatic, while HbE/β° thalassemia can result in transfusion-dependent thalassemia major. Variable quantities of HbA are found in HbE/β⁺ thalassemia cases, and the condition is milder than HbE/β° thalassemia.

HbS/β thalassemia

(See other sickling disorders.)

HbC/β thalassemia

This is restricted to West Africans, some North Africans and southern Mediterranean populations.¹ HbC/β thalassemia is largely asymptomatic and characterized by a mild hemolytic anemia and splenomegaly. A blood smear shows numerous target cells and polychromasia due to the mildly elevated reticulocyte count. Hemoglobin electrophoresis shows a preponderance of HbC and variable quantities of HbA depending on whether it is a β⁺ or β° thalassemia allele. The diagnosis is confirmed by demonstrating HbC trait in one parent and β thalassemia in the other.

The δβ-, γδβ-thalassemias and HPFH syndromes

This group of β-like thalassemia disorders is characterized by a reduced or absent synthesis of β- and δ-globin chains and a variable compensatory increase in γ chain production.⁶⁵ They are much less common than β thalassemia. The distinction between δβ thalassemias and the HPFH syndromes is subtle and originally made on what appeared to be clear-cut clinical and hematological grounds. However, with the elucidation of the molecular basis of these conditions, it became increasingly clear that this broad classification is rather arbitrary, and that there is considerable overlap in many of the parameters that were initially used to differentiate them. It also became clear that the subtle difference between the two subgroups relates to the relatively higher compensatory increase in HbF levels in HPFH compared to the δβ thalassemias.

Heterozygotes for δβ thalassemia have a red cell picture similar to β thalassemia, with hypochromic microcytic red cells, but normal levels of HbA₂ (<3.0%). In addition, however, there is an increased level of HbF (5–15%) that was unevenly distributed (heterocellular) among the erythrocytes. Homozygotes for δβ thalassemia or compound heterozygotes with β thalassemias are not common but have been reported to have clinical phenotypes ranging from mild anemia to thalassemia major.⁶⁶ In contrast, HPFH heterozygotes have essentially normal red cell indices, normal levels of HbA₂ and higher levels of HbF (15–30%) with a homogeneous pancellular distribution of the HbF. HPFH homozygotes are clinically normal, their hemoglobin levels may be increased with mildly hyochromic microcytic red cells. Individuals with HPFH/β thalassemia may have a mild anemia but are clinically asymptomatic.

These conditions are remarkably heterogeneous genotypically; at the molecular level they can be classified into two groups:

The non-deletion mutations are clustered in regions of the promoters that contain binding sites for ubiquitous and erythroid-specific transcription factors. Altered binding of the transcription factors due to the point mutations is thought to be the cause of the elevated HbF levels that range from 5–35% in heterozygotes.

A non-deletion form of δβ thalassemia due to a point mutation inactivating the β gene in cis with a point mutation in the γ gene promoter up-regulating HbF production has also been described.⁶⁷ δβ Thalassemia and HPFH are clearly defined by a significant increase in HbF levels in carriers; the molecular defects are inherited in a Mendelian fashion as alleles of the β-globin complex.

About 10–15% of the healthy adult population have modest increases of HbF (1–5%) in which the inheritance patterns are less clear-cut and traditionally referred to as heterocellular HPFH because the HbF is unevenly distributed among the erythrocytes. Heterocellular HPFH was previously known as Swiss-type HPFH, after the original report in which some members of a group of Swiss soldiers were found to have slight increases in HbF levels.^68,69 It is now clear that heterocellular HPFH represents the high values of HbF and F cells at the upper tail of the common HbF trait distribution.

Although of no consequence to healthy adults, when co-inherited with β thalassemia or sickle cell disease (SCD), heterocellular HPFH leads to increases in HbF that have a major ameliorating effect on the severity of disease.^34,70 Heterocellular HPFH is inherited as a complex trait; multiple genes contribute to the increase in HbF levels. Recent genome wide association studies (GWAS) have mapped three major quantitative trait loci (QTLs) – Xmn1-HBG2, HBS1L-MYB intergenic region (HMIP) on chromosome 6q and BCL11A on chromosome 2p – that account for 50% of the F cell variance in healthy Europeans.^12,13,71 These loci have been shown to impact HbF levels and disease severity in patients from diverse ethnic groups with SCD and β thalassemia.^{37–40,72,73}

εγδβ Thalassemia

These are rare conditions.¹⁰ Affected newborns may present with severe hemolysis and anemia, which is self-limited. In some cases, blood transfusions are necessary during the neonatal period. It is recognized in adults by the hematological phenotypes of β thalassemia trait with normal levels of HbA₂ and HbF. Only heterozygotes have been identified; presumably the homozygous state would not survive early gestation. The molecular defects result from large deletions of the β-globin gene cluster which involve the β-LCR.^65,74–76 The deletions fall into two categories: group I removes all, or a greater part of the complex including the β-globin gene and the β-LCR; group II removes extensive remote upstream regions including the β-LCR but leaving the β-globin gene itself intact, despite which, its expression is silenced because of the absence of the upstream β-LCR. There is no output from the globin genes of the affected cluster. The associated phenotypes of the two groups of deletions are similar.

Genetic basis of disease

Normal individuals have four α-globin genes arranged as linked pairs, α₂ and α₁, at the tip of each chromosome 16, the normal α genotype being written as αα/αα.¹ α Thalassemia most commonly results from deletion of one (−α/) or both (−−/) α genes of the linked pair from chromosome 16, causing a reduction (α⁺) or absence (α°) of α-globin from the affected chromosome, respectively. The homozygous and heterozygous states for α° thalassemia are represented as −−/−− and αα/−−, respectively; while those for α⁺ thalassemia are −α/−α and αα/−α, respectively. As for the β-like genes, full expression of the α-like globin genes is critically dependent on the presence of the major upstream regulatory element (MCS-R2 or HS-40) which lies 40 kb upstream of the cluster. Although more than 50 deletions removing one (−α/) or both (−−/) genes have been characterized, the majority of α thalassemia is caused by six deletions (/−α^3.7, /−α^4.2, /−−^SEA, /−−^MED,/ − ( α)^20.5, and /−−^FIL).¹

The common α° thalassemias are caused by complete or partial deletion of both the structural α-globin genes. These deletions vary in size and are geographically isolated, with two particularly common ones, one in southeast Asia (/−−^SEA) and the other in the Mediterranean region ( /−−^MED). Rarely, α° thalassemias arise from deletions of the upstream α-globin regulatory elements (MCS-R) with the downstream α-globin genes intact but completely inactivated. These deletions are highly variable in size (up to 150 kb have been reported); the smallest of these remove MCS-R1 (HS-48) and MCS-R2 (HS-40), demonstrating the importance of these elements in expression of the α-globin genes.⁷⁸

The molecular basis of α⁺ thalassemia is more complicated; the commonest forms result from deletion of one of the linked pairs of globin genes (/−α^3.7or /−α^4.2).¹ Less commonly, both the α-globin genes are intact, and α⁺ thalassemia results from point mutations (T) that inactivate one of the pair. These point mutations involve the critical sequences that control the various stages of gene expression as encountered in the β thalassemias. To date, 69 causes of non-deletion α thalassemia have been described; 46 of these occur in the dominant α₂ gene (/α^Tα), 17 in the α₁ gene (/αα^T), and the others on a /−α chromosome (/−α^T). In general, the non-deletional α thalassemia determinants have a more severe effect on α-globin output and hematological phenotype than simple deletions that remove one or the other α-globin gene. This may be explained by the majority of mutations affecting the dominant α₂ gene, where expression predominates over the α₁ gene. Another explanation is that, unlike deletional α thalassemia, there appears to be no compensatory increase in expression of the linked functional α gene when the other is inactivated by a point mutation.

A common non-deletion α thalassemia variant in Southeast Asia is Hb Constant Spring (HbCS, /α^CSα) which is due to a single base substitution (TAA→CAA) in the α₂ globin termination codon. This results in readthrough of the 3′ untranslated sequence until another in-phase termination codon is encountered 31 codons later producing an elongated α chain variant of 172 residues, 31 amino acids from the natural Arginine at codon 141. Three other variants (Hb Icaria, HbSeal Rock and Hb Koya Dora) involving different base substitutions in the α₂ termination codon have been identified. Non-deletional α thalassemia can also arise from single base substitutions causing structural α-globin variants that are highly unstable, for example, Hb Quong Sze α125 Leu→Pro (/α^QSα).

Pathophysiology

There is a fundamental difference in the pathophysiology of the α and β thalassemias (Fig. 9.7). Because γ₄ and β₄ tetramers are soluble, they do not precipitate to a significant degree in the bone marrow, i.e. erythropoiesis is more effective than in β thalassemia. However, these β₄ tetramers do precipitate as the red cells age, forming inclusion bodies in the mature erythrocytes, resulting in peripheral hemolysis due to the red cell membrane damage and obstruction in the spleen (Figs 9.7 and 9.8). The degree of anemia and the amount of the tetramers (HbH and Bart’s) produced reflects the severity of the reduction in the output of the α-globin chain.

Fig. 9.7 Diagrammatic representation of the pathophysiology of α thalassemia

Fig. 9.8 Golfball-like appearance of many of the red cells of a case of HbH disease after supravital staining with brilliant cresyl blue. × 1000.

(Courtesy of Professor SN Wickramasinghe).

Genotype-phenotype correlation

Loss of one functioning α gene (αα/−α) is almost completely silent with normal or only slightly hypochromic red cells. Loss of two α genes (−−/αα or −α/−α) produces a mild hypochromic microcytic anemia, the α thalassemia trait. Homozygotes for α° thalassemia (−−/−−) have a lethal condition with intrauterine hemolytic anemia called the Hb Bart’s hydrops fetalis syndrome. Deficiency of α chains gives rise to an excess of γ chains (in fetal life) or β chains (in adult life) which form γ₄-tetramers (Hb Bart’s) and β₄-tetramers (HbH), respectively. The presence of Hb Bart’s or HbH is thus diagnostic of α thalassemia.

HbH disease lies between the two ends of the clinical spectrum, the asymptomatic α thalassemia trait and Hb Bart’s hydrops fetalis. As in β thalassemia intermedia, HbH disease spans a wide range of clinical and hematological phenotypes,^79,80 the diagnostic feature being the presence of HbH inclusions in the peripheral red blood cells (Figs 9.8 and 9.9). The most severe forms can be lethal late in gestation or in the perinatal period (HbH hydrops fetalis).¹ The molecular basis of this order is equally heterogeneous, varying with the geographic distribution of the different α thalassemia variants.^79,80 HbH disease most commonly results from the interaction of α° and deletional α⁺ thalassemia (−−/−α). Less often it can result from the interaction of α° thalassemia with non-deletional forms of α thalassemia (−−/α^Tα) or from homozygous non-deletional α thalassemia (α^Tα/α^Tα). A less severe form of HbH disease in southeast Asia commonly arises from homozygosity or compound heterozygosity for Hb Constant Spring (α^CSα/α ^CSα) or (α^CSα/−−). Very low levels of this elongated α-globin chain (5–8% of the total hemoglobin in homozygotes) are found; the defective α^CS chain production is a consequence of the instability of the α^CS mRNA.⁸¹

Fig. 9.9 Ultrastructure of red cells from a case of HbH disease after incubation with brilliant cresyl blue for 1 h. The HbH-containing cells show membrane-bound, redox-dye-induced masses of denatured HbH. Uranyl acetate and lead citrate. × 6650.

(Courtesy of Professor SN Wickramasinghe).

Diagnosis

Diagnosis is based on the hemoglobin level, red cell indices, examination of the peripheral blood smear and hemoglobin analysis.

Clinical features

The clinical disorders resulting from α thalassemia range from death in utero (Hb Bart’s hydrops syndrome) to a completely silent carrier state.¹ Hemoglobin Bart’s hydrops, caused by homozygosity for α° thalassemia, occurs only in populations where α° thalassemia is common, notably in the Mediterranean and in Southeast Asia. Affected infants are usually stillborn with gross pallor, generalized edema and massive hepatosplenomegaly. The placenta is enlarged and friable and frequently causes obstetric difficulties. All these findings are caused by severe intrauterine anemia. There is no production of α chain, and hence neither fetal nor adult hemoglobin. The hemoglobin consists of approximately 80% Hb Bart’s (γ₄ tetramers) and 20% Hb Portland (ζ₂γ₂). Presumably affected infants survive to term because they continue to produce embryonic hemoglobin. Apart from fetal death, there is a high incidence of toxemia of pregnancy and obstetric complications due to the large placenta.

The intermediate form of α thalassemia is HbH disease, commonly seen in the Mediterranean, Middle East and Southeast Asia. As in β thalassemia intermedia, HbH disease spans a broad spectrum of hematological and clinical severity. Generally, these individuals have a moderately severe anemia and splenomegaly but are usually transfusion independent except during episodes of hemolysis associated with infection, or worsening of the anemia due to progressive hypersplenism. The skeletal deformities and growth retardation characteristic of β thalassemia are not unusually seen. Hemoglobin values range from 7–10 g/dl and the peripheral blood film shows typical thalassemia changes with hypochromia, polychromasia and anisopoikilocytosis; reticulocyte count is normally increased (3–6%). Rarely, the very severe forms of HbH disease can present with hydrops fetalis.

α Thalassemia trait is clinically asymptomatic; the hematological phenotype depends on the number of existing functional α-globin genes. Carriers for α° thalassemia may have very mild hypochromic anemia with red cell indices similar to those of the β thalassemia trait; the MCH is less than 25 pg and the HbA₂ level is normal. Occasional HbH bodies (β₄ tetramers) may be present in the red cell on supravital staining. Deletional α⁺ thalassemia carriers have near-normal hematological findings. The heterozygous states for the non-deletion forms of α⁺ thalassemia are sometimes associated with very mild hypochromic anemia; Hb Constant Spring can be identified by the presence of trace amounts of the variant on hemoglobin electrophoresis at an alkaline pH.

α° Thalassemia carriers can be identified with more certainty in the neonatal period, when they have 5–10% Hb Bart’s (γ₄ tetramers), which disappears over the first few months of life and is replaced by HbH (β₄ tetramers). Some newborn carriers of α⁺ thalassemia have slightly increased levels of Hb Bart’s, in the 1–3% range, but its absence does not exclude the diagnosis.

Background

Sickle cell disease (SCD) was first described by James Herrick from Chicago in 1910 in a student from Grenada.⁸⁴ Presence of peculiar elongated and sickle-shaped red blood cells in the peripheral blood films suggested the term sickle cell anemia. In 1949 Pauling et al demonstrated that this sickling phenomenon was related to an abnormal hemoglobin present in all patients with sickle cell anemia.⁸⁵ Subsequently, in 1956 Ingram showed that the sickle hemoglobin differed from normal adult hemoglobin (HbA, α₂β₂) by the single substitution of glutamic acid to valine at position 6 in the β subunit.⁸⁶

The sickling disorders occur predominantly in black African populations but they are also prevalent throughout the Mediterranean, Middle East and parts of India. Carriers for the β^S gene are protected from Plasmodium falciparum, thus explaining the high gene frequencies in those malarious regions, although the mechanisms underlying such protection are still not clear. The β^S gene in these diverse population groups is caused by the same molecular defect (β codon 6 GAG to GTG), found on four different β haplotypes in Africa, known as the Senegal, Benin, Central African Republic (or Bantu) and the Cameroon types. In addition, it is associated with a different β haplotype (Arab-India) in Saudi Arabian and Asian Indian sickle patients.¹ The evidence suggests multiple independent origins of the β^S mutation although gene conversion on regionally specific β haplotypes cannot be excluded.

Genetic basis and pathophysiology

Apart from the homozygous state for the β^S gene, the syndrome of sickle cell disease (SCD), can also arise from the compound heterozygous state for HbS and β thalassemias (HbS/β thalassemia) and other structural variants such as HbS C (HbSC disease) and D (HbSD) (Table 9.3).

Fundamental to the pathophysiology of sickle cell disease is the polymerization of the sickle hemoglobin (HbS) which is dependent on several factors – concentration of the HbS itself, oxygen saturation, pH, temperature and other factors such as the 2,3-diphosphoglycerate concentration (2,3-DPG) (Fig. 9.10). Fully oxygenated HbS cannot enter the polymer phase whereas partially oxygenated or deoxygenated HbS can.⁸⁷ Mixed hybrids of HbS with non-S hemoglobins, e.g. HbA (α₂ββ^S) and HbC (α₂β^Cβ^S), have a 0.5 probability of entering the polymer phase while those with HbF, i.e. (α₂γβ^S) and HbA₂ (α₂δβ^S), cannot enter the polymer phase.

Fig. 9.10 Pathophysiology of sickle cell anemia.

When a polymer-free solution is deoxygenated, there is a time delay before any polymer is detected. The delay time is exquisitely dependent on the concentration of the deoxy HbS, and also pH and temperature. Delay time shortens as deoxy HbS increases, pH falls and temperature rises. This explains the harmful effects of over-exercise, fever and dehydration. These red blood cells continue to sickle and unsickle, eventually reaching a threshold when sickling becomes irreversible. Although polymerization of HbS is critical, the irreversibly sickled red cells alone are not sufficient for initiating or maintaining the vaso-occlusion that triggers the complications of SCD. Vaso-occlusion is also dependent on factors extrinsic to the cell, such as the state of the vascular endothelium, vascular tone (balance of vasoconstrictors and vasodilators) and activation of platelets and white cells involving cytokines and adhesion molecules. The anemia in SCD is primarily hemolytic, a consequence of the shortened survival of the irreversibly sickled red cells which have a half-life of 20 days compared to a normal RBC life span of 120 days. Survival of the red cells is indirectly related to the level of HbF since red cells containing HbF are less likely to sickle.

Despite the identical sickle mutation, SCD has long been appreciated to exhibit extraordinary phenotypic variability which reflects the genetic background of the individual and the co-inheritance of different modifying genes interacting with the different environmental backgrounds.⁸⁸ Two case reports and a pilot twin study⁸⁹ suggest that environmental factors may be of greater importance in determining clinical expression and complications of SCD. These reports showed that, despite identical β- and α-globin genotypes with similarities in growth, hematological and biochemical parameters, the identical twins had quite different acute pain rate and sickle-related complications.

Nonetheless, genetic factors that influence the primary event of HbS polymerization, such as the causative genotype (HbSS vs. HbSC, HbSβ thalassemia), co-inheritance of α thalassemia and genetic determinants for producing HbF are major predictors of SCD severity. HbF levels are a major predictor of survival and complications in SCD; the risk of early death is inversely associated with HbF levels.⁷⁰ The beneficial effect of HbF is twofold: i) HbF inhibits HbS polymerization, and ii) its presence in the red blood cells reduces the concentration of intracellular HbS. The three recently identified HbF loci have also been shown to impact sickle phenotype.^13,37,73 α Thalassemia which is present in about 30% of SCD patients is associated with less hemolysis, higher hematocrit and hemoglobin levels, lower mean corpuscular volume (MCV) of the red blood cells, and lower reticulocyte counts. The resulting increased hematocrit and blood viscosity may increase certain vaso-occlusive complications such as acute painful episodes, acute chest syndrome and avascular necrosis.

At the secondary level, it is likely that genetic variants controlling differences in vascular hemodynamics, endothelial adhesion, red cell membrane proteins and red cell hydration account for much of the individual variability in vaso-occlusive complications and hemolysis. Genetic studies based on candidate genes have associated several genes with some complications of SCD including stroke, leg ulcers, priapism and avascular necrosis.⁹⁰ However, candidate genes were selected based on our understanding of the sickle pathophysiology. Genome wide association studies (GWAS) that permit a hypothesis-free and unbiased assessment of all genes should enlighten us on the key pathways and molecular mechanisms underlying the sickle pathophysiology. For such a complex disease, GWAS will require large numbers (thousands) of well characterized patients.

Diagnosis

Diagnosis is established by a combination of several tests for the detection and quantification of HbS plus the other Hbs for the elucidation of the different genetic interactions: hemoglobin electrophoresis on cellulose acetate (pH 8.6) and agar (pH 6.2) and positive sickling or solubility tests.⁹¹ In sickle cell anemia (HbSS), it is usual to see sickled cells, Howell–Jolly bodies and changes typical of hyposplenism. In HbSC disease target cells are prominent and HbC crystals may be present and in HbSβ thalassemia (or HbS with α thalassemia) hypochromic microcytic target cells are present.

Because the gene for HbS (and the structural hemoglobin variants, HbC and HbE) and the thalassemias (both α and β) occur together at high frequency in many populations, it is not uncommon for an individual to inherit genes for both types of condition, posing difficulties in diagnosis. Although these diagnostic difficulties can normally be resolved by family studies, often family members may not be accessible and one has to rely on a careful assessment of the hemoglobin electrophoresis and the red cell indices. In sickle cell trait (Table 9.3) uncomplicated by α or β thalassemia the level of HbS varies between 40% and 45% and is always less than 50%. This is because the β^S chain is positively charged and is less able to compete with the negatively charged β^A chains for the positively charged α-globin subunits.^92,93 Carriers for α thalassemia and HbS have lower HbS levels, varying from ~25% (in −α/−α) to ~35% (in αα/−α). Electrophoretically there may be little difference between HbSS with high levels of HbF and HbS/β° thalassemia but the latter is accompanied by hypochromic microcytic red cells and an elevated HbA₂ level. These features are also present in HbS/β⁺ thalassemia, but in this case, there is some HbA and the HbS is, of course, more than 50%. However, increased levels of HbA₂ do not always reflect the co-existence of β thalassemia. In terms of electrostatic attraction for the α globin subunit, the δ globin subunit lies between that of β^A and β^S. Since the δ globin chain has a greater affinity for the α subunit compared to β^S, co-inheritance of α thalassemia with HbS can mimic HbS/β thalassemia. In both scenarios, the HbS level is lower than expected, the red cells hypochromic microcytic and the level of HbA₂ increased.

Clinical features

Sickle cell trait (carrier status) is a benign condition and generally does not cause any clinical disability. However, under certain extreme conditions, such as severe pneumonia, flying in unpressurized aircraft or rigorous physical exercise with ensuing dehydration, vaso-occlusive episodes can occur.^94,95

SCD causes a chronic debilitating illness characterized by hemolytic anemia interrupted by episodic acute pain and acute clinical events resulting in end-organ damage and a reduced life span. A main determinant of disease severity is the causal genotype. Individuals homozygous for the β^S gene (HbSS, sometimes referred to as sickle cell anemia) and individuals with HbSβ° thalassemia, whose hemoglobin composition is almost all HbS, tend to have the most severe disease, followed by HbSC and HbS β⁺ thalassemia. Compared to HbSS, HbSC patients have less hemolysis and thus are less anemic. All complications that occur in HbSS patients have also occurred in HbSC patients although at a lower frequency and they appear at a later age. Complications related to blood viscosity and microvascular occlusions such as avascular necrosis and proliferative retinopathy are just as frequent in HbSC patients. However, even within identical genotypic groups, the clinical course is remarkably variable; at one extreme are the completely asymptomatic patients with SCD discovered by chance on routine hematological examination such as during antenatal screening and at the other are patients who present with dactylitis in early childhood, followed by recurrent hospital admissions due to frequent acute pain. Most patients fall between the two extremes and are able to lead a relatively normal life. All SCD patients have a shortened life span; the life span of HbSC patients is shortened when compared to control populations but is longer than that of HbSS patients.

Sickle cell anemia (HbSS) normally presents in infancy after 6 months of life with attacks of dactylitis, manifested as painful swelling of the fingers and feet, the so-called ‘hand-foot syndrome’. At this stage, the infant is usually anemic with mild jaundice and the spleen palpable. Splenomegaly usually resolves due to repeated infarctions of the spleen, the ‘autosplenectomy’ manifested by typical post-splenectomy changes in the peripheral blood film, visible as early as 10–12 months of age. Typically, these children have a chronic hemolytic anemia with a hemoglobin level that varies between 6 and 8 g/dl and a reticulocyte count of 10–20%. Serum bilirubin levels are elevated and peripheral blood smear shows polychromasia and poikilocytosis with a variable number of sickled erythrocytes.

The chronic hemolysis of sickle cell anemia is punctuated by acute clinical events termed sickling crises traditionally classified as vaso-occlusive, sequestration, aplastic or hemolytic, but in reality each acute clinical event has varying components of the different crises. The most common are the acute painful episodes due predominantly to vaso-occlusion in localized areas of bone marrow leading to infarction. Commonly affected areas are long bones, ribs, sternum and lumbosacral spine; often multiple sites are affected. The patient experiences a rapid onset of deep, throbbing bone pain in the affected areas, usually without physical findings but sometimes accompanied by local tenderness, warmth and swelling. Marrow aspirated from areas of bone tenderness have revealed infarction of the marrow tissue. Occasionally, abdominal pain is the major symptom and can pose a difficult problem in differential diagnosis. The abdominal crisis is accompanied by distension and rigidity with loss of bowel sounds, findings typical of an acute surgical abdomen. Acute cholecystitis and gallstones should also be considered.

Acute chest syndrome is characterized by acute dyspnea, pleuritic pain and fever, accompanied by a new pulmonary infiltrate on chest X-ray. It is often accompanied by a significant fall in the hemocrit which may reflect sequestration of the sickled cells in the pulmonary vessels. The distinction from pulmonary infection is often very difficult, particularly as infection and infarction usually co-exist. Acute chest syndrome is a frequent cause of admission but can be triggered by acute pain; it is the commonest cause of death in young patients.⁷⁰ The brain is the major site of morbidity in children; acute vaso-occlusion in the CNS usually presents in childhood, either as fits, transient neurological symptoms resembling ischemia attacks, or with a fully developed stroke.⁹⁶ Overt stroke affects about 6% of 10-year-olds with a peak between the ages of 2 and 5 years (incidence of 1.02 per 100 patient years), being rare in infants less than 1 year of age. Recurrent attacks are common; 70% of patients experience a recurrence within 3 years and many are left with permanent motor and intellectual disabilities. Long-term/lifetime blood transfusion is recommended for secondary prevention of stroke. Children with HbSS are at highest risk followed by HbS/β° thalassemia and HbSC disease. Priapism, defined as a sustained, painful and unwanted erection, is a well recognized complication in young men with SCD. It results from vaso-occlusion of the outflow vessels from the corpora cavernosa by sickled erythrocytes. This complication may present as multiple short-lived episodes (’stuttering’ priapism) which may progress to ‘severe prolonged’ priapism lasting several days and leading to permanent penile dysfunction.

Transient marrow aplasia can have a profound effect with reticulocytopenia and a very sudden drop in hematocrit. These aplastic crises appear to result from intercurrent infections, particularly due to human parvovirus B19, and often occur in epidemics, frequently involving more than one sibling in the same family.

Sequestration crises commonly involve the spleen in the first two years of life. Definition of splenic sequestration includes a sudden, massive enlargement of the spleen concomitant with a drop in hemoglobin concentration (≥20 g/l) with substantial reticulocytosis. As the crisis progresses, a large proportion of the circulating red cell mass may be trapped in the spleen leading to profound anemia and death. Splenic sequestration shows a tendency to recur in the same individual and splenectomy is often recommended after a recurrence. A similar type of sequestration may occur in the liver in adult life, causing a dramatic fall in the hematocrit.

Patients with sickle cell disease are particularly susceptible to infection, due to Streptococcus pneumoniae, Salmonella, Escherichia. coli and Hemophilus influenzae. Osteomyelitis is common and results from infection of bone infarcts. Pneumococcal pneumonia and overwhelming septicemia are particularly important causes of death in infancy and childhood because of hyposplenism.

Repeated vaso-occlusive events and vasculopathy related to the chronic hemolysis ultimately result in end-organ damage and almost any organ can be affected. An observational study noted that by the fifth decade, ~50% of patients would have one or more of sickle-related end-organ damage.⁹⁷ Avascular necrosis of the femoral head may lead to total disability, frequently requiring a total hip replacement. Virtually every patient with sickle cell anemia has some form of renal impairment. Sickling of the erythrocytes is enhanced in the hypertonic, hypoxic and acidotic environment of the renal medulla leading to progressive infarction of the medullary papillae. There is progressive inability to concentrate urine, polyuria, nocturia and enuresis, which is common in children. Eventually the glomerular damage causes end-stage renal failure, particularly in patients over 40 years of age. Due to the chronic hemolysis, pigment gallstones are very common and are seen in one third of SS patients by the age of 10 years. However, it is difficult to assess its clinical significance as not all patients develop clear-cut cholecystitis. Gilbert’s disease, which is present in about 30% of Africans, is a predisposing factor. Recurrent chronic leg ulceration is common and can be a major handicap (Fig. 9.11). The lesions normally occur just above the medial or lateral malleoli and seem to be more common in those patients with severe anemia. Proliferative retinopathy leading to progressive visual loss is an important ocular complication although this is more common in HbSC disease.

Fig. 9.11 Chronic leg ulcer in an adult with sickle cell anemia. Note the increased pigmentation of the skin around the ulcer.

(Courtesy of Professor SN Wickramasinghe).

Patients with SCD have a shortened life expectancy. Among HbSS patients in Jamaica, a life expectancy of 58 years for men and 66 years for women has been estimated, while in the United States a median age of death of 42 years for men and 48 years for women was reported in 1994.

Management

Bone marrow transplantation offers the only hope of cure for SCD, available and appropriate for a minority of patients. For the majority of SCD patients, care remains primarily reactive and preventive. Two non-transplant options have a major impact on management of SCD – blood transfusion and hydroxyurea therapy.

In general, management requires a multidisciplinary team effort and consists of continuous general medical care, attention to good nutrition, immunization, avoidance of extremes of temperature and dehydration, and treatment of complications as they arise.⁹⁸ In untreated patients, mortality is highest in the first year of life. In high risk populations, neonatal screening programs should be established to identify affected babies which should be referred to a specialist center and a comprehensive care plan initiated as soon as possible. These babies should be started on prophylactic penicillin at 3–4 months of age followed by polyvalent pneumococcal vaccine at the age of 6–12 months. There should be a systematic approach to education of parents (and later on, the child) on recognition of the different acute clinical events and signs of illness that play a critical role in successful management of children with SCD.

Most patients with SCD generally tolerate the relatively low Hb levels and blood transfusion is usually not required. Over the last decade, however, blood transfusion therapy is increasingly used in SCD with an ever expanding list of indications. Transfusion of normal blood provides benefit by correcting the low oxygen-carrying capacity as well as improving microvascular circulation by decreasing the population of sickled red cells. Regular blood transfusion also suppresses endogenous erythropoiesis and production of HbS. The choice of transfusion therapy – simple top-up, automated exchange (erythrocytapheresis), and partial exchange transfusion – depends on the needs of the patient. Except for severe anemia, exchange transfusion offers many advantages, and is increasingly used. Top-up transfusion is indicated when there is a sharp fall in the hemoglobin level due to bone marrow failure (aplastic crisis) or increased hemolysis (see Table 9.4 for indications for transfusion therapy in SCD).

Table 9.4 Indications for blood transfusion in sickle cell disease