Abnormalities of the structure and synthesis of hemoglobin

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CHAPTER 9 Abnormalities of the structure and synthesis of hemoglobin

Normal human hemoglobin: structure and synthesis

The switch from embryonic to fetal hemoglobin production begins as early as week 5 of gestation and is completed by week 10 (Fig. 9.1). β-globin expression starts as early as week 8 but the synthesis remains low, increases to approximately 10% at weeks 30–35 of gestation with a dramatic up-regulation of β-globin synthesis just before birth, coinciding with a precipitous decline in γ-globin expression. At birth, HbF (α2γ2) comprises 60–80% of the total hemoglobin, falling to ~5% at 6 months of age and eventually reaching the adult level of about 1% at 2 years, by which stage mutations affecting the adult β-globin gene should become apparent. The switch from fetal to adult hemoglobin production is not total; γ-globin production persists at a low level throughout adult life, with the residual amounts of HbF restricted to a small subset (0.2–7%) of erythrocytes termed F cells.1

Each of the α-like and β-like globin chains are encoded by genetically distinct gene clusters, the α-like cluster on the tip of chromosome 16p (5′-ζ-α21-3′), and the β-like cluster on chromosome 11p15.5 (5′-ε-Gγ-Aγ-δ-β-3′) (Fig. 9.1). In both clusters, the genes are arranged along the chromosome in the order in which they are expressed during development, suggesting that gene order may be important in the program of their expression. The coding region of each globin gene is interrupted by two intervening sequences (IVSs) or introns. In the β-like globin genes, IVS-1 (122–130 bp) interrupts the sequence between codons 30 and 31, and IVS-2 (850–900 bp), between codons 104 and 105. In the α-like globin genes, IVS-1 interrupts the coding region between codons 30 and 31, and IVS-2, between codons 99 and 100. In addition to the primary cis-determinants of individual globin gene expression which are found in the promoter region immediately upstream of each gene, there are other local regulatory elements known as enhancers which are located at variable distances from the individual genes.1

Throughout development, the appropriate genes of the two globin gene clusters are coordinately expressed, maintaining a tight balance in the production of α- and β-like globins needed for the synthesis of normal hemoglobin; imbalance between the different proteins may lead to anemia. Expression of the individual genes within each cluster is controlled by complex interactions between the local regulatory sequences within each gene and regulatory elements upstream of the cluster, mediated by a series of transcription factors. In the β cluster, the upstream element is referred to as the β locus control region (β-LCR), which consists of five DNase I hypersensitive sites (designated HSs1–5) distributed between 5 and 25 kb upstream of the ε-globin gene (Fig. 9.1). The region was originally implicated in patients with anemia due to β-globin chain deficiency despite carrying normal β-globin genes. These patients were subsequently shown to be carrying mutations that deleted sequences upstream of the ε-globin gene. These natural mutants prompted experiments that demonstrated the absolute importance of the β-LCR for high levels of globin gene expression and led to a general definition of LCRs: elements that confer high levels of tissue-specific expression to a cis-linked gene dependent on copy-number but independent of position of integration site. The corresponding region in the α globin cluster consists of the four multispecies conserved sequence regions (MCS-Rs), lying 30–70 kb upstream of the α-globin genes termed MCS-R1 to R4.2 Of these elements, only MCS-R2, which consists of a single DNase hypersensitive site, has been shown to be essential for α-globin expression. MCS-R2, which lies 40 kb upstream of the cluster, is also known as HS-40. The β-LCR establishes a transcriptionally active chromatin domain that encompasses the whole β-globin cluster and acts as a unique enhancer while the α-globin HS-40 is most similar to HS2 of the β-LCR and acts as an enhancer. In both clusters, full expression of the downstream genes is critically dependent on the presence of the upstream regulatory elements.

The precise molecular mechanisms by which the globin genes are expressed in a tissue- and developmental-stage-specific manner are still poorly understood. Each cluster contains various binding sites for both erythroid-specific and more ubiquitous DNA-binding proteins in the upstream regulatory elements as well as in the local promoters of the genes. Tissue-specific expression may be explained by the presence of the binding sites for the erythroid-specific transcription factors. These motifs include (A/T) GATA (A/G) binding the tissue-restricted zinc finger proteins (GATA-1 and GATA-2), and their cofactors (FOG1 and FOG2); ‘CACCC’ binding the erythroid Krüppel-like factors (EKLF and FLKL) and, ‘TGA(C/G)TCA’ (NF-E2/AP-1 like) elements, binding the b-Zip family of proteins (NF-E2, Nrf1, Nrf2, Nrf3, Bach1 and Bach2).1,3 These transcription factors can both activate and repress gene expression. They are modular proteins and their dual functions are mediated via the distinct domains either through direct DNA binding or interaction with other proteins. Recent work has also shown that part of the function of these transcription factors involves modification of chromatin states. It seems likely that these erythroid-specific transcription factors form part of a network of factors that commit hemopoietic cells to erythroid differentiation. Elucidation of the complexity of transcription factor function has been facilitated in recent years by a series of technological advancements that included ‘chromosome conformation capture’ (3C, 4C and 3C-sequencing), RNA TRAP (tagging and recovery of associated proteins), chromatin immunoprecipitation (ChIP), and microarray analysis to identify gene targets of the transcription factors.4 The 3C technique can be used to demonstrate interactions among chromosomal fragments and indeed, showed that the β-LCR and actively transcribed globin genes are in close spatial proximity in a cluster referred to as an active chromatin hub (ACH).5

The mechanisms by which developmental regulation is controlled are less clear and rely on two mechanisms, autonomous gene silencing and gene competition.6 It appears that the ε and ζ genes are switched on in embryonic cells and autonomously switched off in definitive cells (liver and bone marrow) in which they cannot be substantially reactivated. The regulatory sequences mediating silencing of the ε-globin gene have been mapped to its distal and proximal promoters. The second switch from γ to β gene expression is more complex and involves both autonomous silencing of the γ genes and competition between the γ and β genes for the β-LCR. The LCR up-regulates only one gene at a time, and the genes compete with each other for activation by the LCR.7 The main determinant for activation of a gene is the relative distance from the LCR. In human adult tissues, the switch leading to expression of the further downstream located β and δ genes is achieved by autonomous silencing of the fetal γ genes. The balance between the γ- and β-gene expression is thought to be mediated by changes in the repertoire and/or abundance of various nuclear factors favoring particular promoter–LCR interactions. A variety of nuclear factors involved in the transcriptional regulation and hemoglobin switching have been suggested, including BCL11A, SOX6, GATA-1, KLF1, NF-E4, COUP-TF, DRED /TR2 /TR4, Ikaros-PYR and BRG1 (SW1/SNF).8 KLF1 (Krüppel-like factor 1), also known as erythroid Krüppel-like factor (EKLF) is restricted mainly to erythroid cells; it is also a highly promoter-specific activator, binding with high affinity to the β-globin CACCC box.9 Mutations found in β-globin gene CACCC box abrogate EKLF binding result in reduced β-globin expression and β thalassemia.10

BCL11A (B cell lymphoma/leukemia 11A), also known as Evi9, CTIP1, is a zinc finger transcriptional factor, essential for normal lymphoid development.11 Its role in hemoglobin switching was originally implicated in genetic association studies on HbF levels in humans.12,13 Subsequent collective clinical and functional studies suggest that BCL11A acts as a stage-specific repressor of γ-globin expression.14,15

Hemoglobin switching has been the subject of intensive investigation for two main reasons: 1) the globin system provides an excellent model for understanding developmental gene regulation, and 2) understanding how switching is controlled is expected to lead to the development of strategies for the treatment of hemoglobinopathies. Thus, BCL11A appears as an attractive target for reactivation of HbF in patients with β thalassemia or sickle cell disease.

Inherited disorders of hemoglobin

The vast majority of disorders affecting hemoglobin are inherited; it is estimated that ~7% of the world’s population are carriers for different inherited disorders of hemoglobin, making them the commonest monogenic diseases.16 The disorders can be divided into two main groups, those in which there is a structural change in a globin chain (hemoglobin variants) and the thalassemias, which result from a quantitative deficiency in one or more of the globin chains of hemoglobin. Hemoglobin variants cause a wide range of clinical problems including sickle cell disease, unstable hemoglobin, decreased oxygen affinity and increased oxygen affinity, but the majority of hemoglobin variants cause no significant change in hemoglobin properties or clinical problems. Some hemoglobin variants are synthesized at a reduced rate, resulting in a phenotype of thalassemia. The most common example is HbE, β26 (Glu→Lys), in which the substitution at β-codon 26 (GAG→AAG) that causes HbE also causes alternative splicing of the β-globin mRNA, leading to a reduction of the normally spliced β message that encodes the variant. Other hemoglobin variants result in a thalassemic phenotype due to its extreme instability leading to a functional deficiency of the globin chain variants; for example, Hb Geneva, a dominantly inherited β thalassemia,17 and Hb Constant Spring (an α thalassemia variant).1 Some hemoglobin disorders are acquired; these can also be classified into those characterized by a reduced synthesis of the globin chain (e.g. acquired HbH disease) and those which alter the structure and function of hemoglobin so that oxygen transport is affected (e.g. carboxyhemoglobinemia, methemoglobinemia).1

There is another group of β-thalassemia-like disorders referred to as δβ thalassemias and hereditary persistence of fetal hemoglobin (HPFH). These are caused by mutations that alter the switch from fetal to adult hemoglobin, and are distinguishable from β thalassemias by the substantial increases in HbF levels.1

Thalassemias

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

β Thalassemias

The β thalassemias pose by far the most important public health problems because they are common and usually produce severe anemia in individuals who have inherited two copies of the β thalassemia gene.

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.

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.

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 origin31,32 (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.

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 HbA22δ2) 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 cases3436 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

I Homozygous or compound heterozygous state for β thalassemia
  (a) Inheritance of mild β thalassemia alleles, β ‘silent’ and β++, in homozygous or compound heterozygous states
  Phenotype depends on the sum total of β-globin output
  (b) Co-inheritance of α thalassemia
  Phenotype depends on severity of imbalance between α/non-α globin reflecting severity of α and β-globin deficit
  (c) Increased HbF response
  – β-globin gene promoter mutations (deletional or non-deletional)
  – Co-inheritance of HbF quantitative trait loci (QTLs) for HbF on 6q (HBS1LMYB intergenic polymorphisms, BCL11A on 2p, Xmn1-Gγ on HBB cluster)
II Heterozygous state for β thalassemia
  (a) Co-inheritance of extra α globin genes
  (ααα/αα,ααα/ααα,αααα/αα,αααα/αααα, and segmental duplication of whole α-globin gene cluster)
  (b) Dominantly inherited β thalassemia
  (Hyperunstable β-globin chain variants)
  (c) Somatic deletion of the other β-globin locus – mosaicism
III Compound heterozygotes for β thalassemia and β chain variants
  e.g. HbE/β thalassemia
IV Compound heterozygotes for β thalassemia and HPFH or δβ thalassemia
  A considerable variation in clinical phenotype of these genetic interactions has been observed

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.5153

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.

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.

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