Thalassemias and Related Disorders: Quantitative Disorders of Hemoglobin Synthesis
Caterina Borgna-Pignatti
Renzo Galanello*
Dedication: We wish to dedicate this chapter to Rino Vullo and Antonio Cao who passed away on June 22, 2010, and June 21, 2012, respectively.
Rino Vullo in Ferrara and Antonio Cao in Cagliari pioneered a new way of approaching thalassemia, believing in the power of science to change the fate of what was considered for many decades a hopeless disease. Their scientific and human contributions will remain in the history of hematology.
*Dr. Renzo Galanello died on May 13, 2013. He was a pediatrician, a hematologist and a geneticist of enormous competence. We will miss his enthusiastic approach to science, to medicine, to life itself.
The thalassemias are a group of congenital anemias that have in common deficient synthesis of one or more of the globin subunits of the normal human hemoglobins (Hbs). The primary defect is usually quantitative, consisting of the reduced or absent synthesis of normal globin chains, but there are mutations resulting in structural variants produced at reduced rate (e.g., HbE, Hb Lepore) and mutations producing hyperunstable hemoglobin variants with a thalassemia phenotype (thalassemic hemoglobinopathies). Therefore, a rigid differentiation from the qualitative changes of hemoglobin structure that characterize the hemoglobinopathies is no longer appropriate. According to the chain whose synthesis is impaired, the most common thalassemias are called α-, β-, γ-, or δβ-thalassemias. These subgroups have in common an imbalanced globin synthesis, with the consequence that the globin produced in excess is responsible for ineffective erythropoiesis (intramedullary destruction of erythroid precursors) and hemolysis (peripheral destruction of red cells). In the last few years, the advances of DNA analysis have permitted understanding the basic aspects of gene structure and function and the characterization of the molecular basis for deficient globin synthesis. The thalassemias result from the effect of a large number of different molecular defects, which may interact, leading to a variety of clinical and hematologic phenotypes.
PREVALENCE, GEOGRAPHIC DISTRIBUTION, AND THE ROLE OF MALARIA
Thalassemia is considered one of the most common genetic disorders worldwide. It occurs in a particularly high frequency in a broad belt extending from the Mediterranean basin through the Middle East, Indian subcontinent, Burma, Southeast Asia, Melanesia, and the islands of the Pacific. According to data collected through the Hereditary Disease Program of the World Health Organization and based on local surveys and reports by visiting experts, the carriers of hemoglobin disorders in the world are estimated to be 269 million.1 Recent global epidemiologic data on the demographics and prevalence of hemoglobinopathies have established that these disorders represent a significant health problem in 71% of 229 countries and that around 1.1% of couples worldwide are at risk for having children with a hemoglobin disorder.2 From data available it has been estimated that the annual number of births is 22,989 for β-thalassemia major; 5,183 for Hb Bart hydrops fetalis syndrome; 9,568 for hemoglobin H disease (the intermediate form of α-thalassemia); and 19,128 for HbE/β-thalassemia.2, 3
Diseases caused by α-thalassemia are encountered commonly in Southeast Asia and China with up to 40% of the regional population being carriers, and less commonly in India, Kuwait, the Middle East, Greece, Italy, and Northern Europe.4 Southeast Asia is the area of the world where the frequency of α-thalassemia is so high as to cause a major public health problem, because of the increased number of patients with severe HbH disease and fetuses with Hb Bart hydrops fetalis. In the eastern oasis of Saudi Arabia, more than 50% of the population appears to have a clinically silent form of α-thalassemia, and HbH disease is recognized with increasing frequency.5 In a random population sample, the gene frequency of deletion-type α-thalassemia-2 (-α) was 0.18 in Sardinians and 0.07 in Greek Cypriots; the occurrence of nondeletion α-thalassemia is estimated to be one-third that of the deletion-type.6 In African-Americans, α-thalassemia is relatively common, but rarely is it of clinical significance. Of 211 healthy African-Americans in whom the α-globin genotype was characterized, 27.5% lacked a single α-globin gene, and 1.9% lacked two of the four α-globin genes.7 With the rise in Asian immigration to North America over the past few decades the prevalence of α-thalassemia and other hemoglobinopathies has increased steadily.8 The epidemiologic changes in the prevalence of hemoglobin disorders have important implications for public health programs, including new laboratory strategies, newborn screening, counseling, and patient management.9, 10 About 3% of the world’s population (150 million people) carry β-thalassemia genes. In Europe they are particularly prevalent in inhabitants of Italy and Greece. In Italy, the highest prevalence of the carrier state, in descending order, has been found in Sardinia (10.3%), the Delta region of the Po River near Ferrara (8%), and Sicily (5.9% with an almost equal distribution over the entire island).11, 12, 13 In Greece, the prevalence varies considerably, ranging from <5% to nearly 15% in the southern and central areas.32, 33 In Cyprus, one individual in seven is a carrier of β-thalassemia and one individual in 1,000 is currently homozygous.1 In Sardinia the incidence of homozygous β-thalassemia is 1 in 250 live births. There are an estimated 3,500 individuals with thalassemia major in Greece and 4,300 in Italy14 (Cianciulli personal communication). β-Thalassemia is encountered less often in the northern and western parts of Africa. In the Maghreb (African countries opening on the Mediterranean) frequencies vary from 3% in Algeria to 7% in Morocco and Lybia.15 In Egypt, thalassemia represents a serious health problem, with a predicted 1,000 new patients born each year.16 In Turkey, the frequency varies from 0.8% to 10.8%.17 It has been described in high frequencies (10% to 20%) in Indian and Kurdish Jews.18 In Arabs it averages 2%.19 Few data are available for Pakistan.20 Among Indians, frequencies between 3.5% and 14.9% have been reported.21 In North America, thalassemia used to affect mainly individuals of Mediterranean origin and African-Americans, but is, at present, most frequently observed in Asians. In fact, increases in Asian immigration and births in the United States, particularly in California, have led to a prevalence rate of HbE/β-thalassemia among Southeast Asians of ˜1 in 2,200.22 In one survey of healthy black men, heterozygous β-thalassemia was documented in 1.4%.23 β-Thalassemia in Jamaica may have its origin in both the African and Asian immigrant populations.24 In sporadic cases, β-thalassemia has been noted in Northern Europeans with no apparent Mediterranean or Asian ancestry.15 Although well documented in natives of Southeast Asia and southern China, β-thalassemia is far less prevalent in these regions than is α-thalassemia.25 HbE, the hallmark of Southeast Asia, is most frequently found at the border of Thailand, Laos, and Cambodia, where the frequencies may reach 50% to 60%. In Great Britain, which today is a multiethnic society due to substantial
migration from Cyprus, the Indian subcontinent, Southeast Asia, and the Middle East, it is estimated that 0.37 per 1,000 fetuses have a major hemoglobin disorder, 20% being thalassemias and 80% being sickle cell disease. A formal patient register was also recently established in Great Britain in 1997. At the end of 1999, 807 patients with thalassemia major were alive and residing in the United Kingdom, most of whom were of Pakistani or of Cypriot origin.26 Detailed information on the frequency of thalassemia in different world regions is available from Modell and Darlison,2 and Galanello et al.27
migration from Cyprus, the Indian subcontinent, Southeast Asia, and the Middle East, it is estimated that 0.37 per 1,000 fetuses have a major hemoglobin disorder, 20% being thalassemias and 80% being sickle cell disease. A formal patient register was also recently established in Great Britain in 1997. At the end of 1999, 807 patients with thalassemia major were alive and residing in the United Kingdom, most of whom were of Pakistani or of Cypriot origin.26 Detailed information on the frequency of thalassemia in different world regions is available from Modell and Darlison,2 and Galanello et al.27
The Role of Malaria
Even in the first description from Italy of children affected by thalassemia, Maccanti observed that all the patients came from malarial areas and, 20 years later, Vezzoso noted that the distribution of Cooley’s anemia in Italy coincided with that of malaria.28, 29 The hypothesis that malaria had an influence in maintaining the high prevalence of hemoglobinopathies in the world was first suggested in 1949 by Haldane, who also proposed that the small red cells of the carriers of thalassemia could be more resistant to the malaria parasites.30 A few years earlier, Neel and Valentine had calculated that, in the absence of some kind of selective pressure, the mutation rate for thalassemia had to be in the order of 1 in 2,500.31 Carcassi et al. and Siniscalco et al. in Sardinia obtained suggestive epidemiologic data on the distribution of thalassemia and malaria, but their results were not confirmed in other populations.32 Molecular biology studies helped to clarify at least some aspects of the problem. In fact, they revealed the very high number of β-thalassemia mutations that are, for the most part, regionally specific, as well as the association of particular mutations with specific β-globin gene haplotypes. The regional specificity of mutations suggests local processes for their elevation to high frequencies, while the close association with specific haplotypes suggests a recent cause. These observations point to the conclusion that the selective pressure of malaria has amplified the β-thalassemia genes to high frequency so recently that neither migration, recombination, nor genetic drift could have had sufficient time to bring them into spatial or genetic equilibrium with their background.33
The mechanism by which the thalassemia heterozygote could be protected from malaria is still not clear. Several studies have demonstrated reduced red cell invasion by malaria parasites in the severe forms of thalassemia, but the results in the heterozygous states for α– and β-thalassemia have been contradictory.34, 35, 36 A provocative study performed in newborns with α-thalassemia revealed an increased susceptibility to infection by Plasmodium vivax, a less severe form of malaria, and suggested that this could confer permanent cross-species protection against Plasmodium falciparum.37 Both parasites invade preferentially young circulating red cells, and early infection in a period of life when maternal antibodies are still present could protect them from later severe disease. Also, some form of immunologic mechanism could be involved in protecting carriers of thalassemia from malaria and possibly from other diseases as well.38 However, recent in vitro studies using red blood cells (RBCs) with common hemoglobinopathies (e.g., α– and β-thalassemias, HbS, HbC, HbE) and enzyme (glucose-6-phosphate dehydrogenase [G6PD]) defects have shown a reduced parasite invasion/growth and an increased susceptibility to phagocytosis of the infected RBC as a malaria-protective effect.39, 40 A recent systematic review and meta-analysis of studies that estimated the risk of malaria in patients with and without hemoglobinopathies, showed a decreased risk of severe P. falciparum malaria in sickle cell carriers, homozygous and heterozygous hemoglobin C, and homozygous and heterozygous α-thalassemia.41 These hemoglobinopathies differ substantially in the degree of protection provided and confer mild or no protection against uncomplicated malaria and asymptomatic parasitemia.
Moreover, evidence has been obtained for complex epistatic interactions among different inherited hemoglobin disorders with respect to malaria protection, at least partly explaining some profound differences in their distribution among different populations.3
GENETIC MECHANISMS AND MOLECULAR PATHOLOGY
Synthesis of hemoglobin, the molecule used for oxygen transport, is directed by two gene clusters: The α locus, which contains the embryonic ζ gene, plus the two adult α genes; and the β cluster, which contains the embryonic ζ, the fetal Gγ and Aγ, and the adult δ and β genes (Fig. 34.1). Different hemoglobins are produced during development and two globin gene switches take place: the embryonic to fetal switch (ε to γ and ζ to α), which starts very early in pregnancy and is completed at 10 weeks of gestation; and the fetal to adult switch (γ to β), which occurs during the perinatal period.42 The globin gene switches, besides the changes in hemoglobin composition, come with changes in other morphologic and biochemical characteristics of the erythropoietic cell line, including the shift from the nucleated megaloblast to macrocyte and to the definitive normocyte; the shift in the site of erythropoiesis from the yolk sac to liver, spleen, and bone marrow; and changes in the membrane antigenic profile and in the red cell glycolytic activity.43 Recent discoveries about transcriptional regulation of fetal hemoglobin represent a major advance in understanding the switching mechanisms and for developing targeted approaches to ameliorate the severity of β-hemoglobinopathies. Genetic linkage and genome-wide association studies have led to the identification of a variety of nuclear factors acting as multiprotein complexes, involved in globin gene regulation and switching.44, 45, 46, 47, 48, 49 The most relevant are BCL11A and KLF1. BCL11A is a zinc-finger transcription factor necessary for normal B and T lymphopoiesis, that in cooperation with other transcriptional factors, acts also as a direct developmental stage-specific repressor of fetal hemoglobin expression.50 KLF1 is a zinc-finger erythroid-specific transcriptional regulator, known as an activator of the β-globin gene through direct binding to the critical CACCC box promoter element, that activates BCL11A expression by associating with the BCL11A promoter.48, 51 Therefore, KLF1 has a dual role in globin gene regulation, functioning as a direct activator of the β-globin gene and an indirect repressor of the γ-globin gene.52, 53, 54 Protein-protein and protein-DNA interactions, involving the β-globin gene cluster and the locus controlling region (LCR), result in complex developmental-related chromatin modifications that activate or repress the different globin genes (Fig. 34.2). The globin genes are relatively small and composed of three exons, coding for functional domains of the hemoglobin, and two intervening sequences (introns).43 The different globin gene expression during development is controlled through the action of transcription factors and regulatory elements (promoters, enhancers, and silencers) that flank each globin gene, and of more remote sequences important for the regulation of all of the cluster (see below). The promoter of each globin gene contains sequences that act as binding sites for erythroid-restricted or ubiquitously expressed transcription factors responsible for tissue-specific and developmental-specific regulation of the globin genes. Particularly relevant promoter sequences are the TATA box, situated 30 bp upstream of the initiation site, and the CAAT and CACCC boxes at approximately -70 and -110 bp from the initiation site, respectively. Among the transcription factors, some have been studied in detail for their role in the regulation of globin gene expression. GATA-1 is the first of a family of DNA-binding proteins, whose binding sites are present in one or more copies in almost all the regulatory elements of the globin genes.55 Nuclear factor-erythroid 2 (NFE2), EKLF, FOG-1, and SP1 are other transcription factors involved in the expression
of the β-globin gene.56, 57, 58, 59 Like other genes, globin genes possess a series of motifs critical for their expression: the CAP site, which indicates the start of transcription; the AGT initiation codon, which is the signal for starting translation in messenger RNA (mRNA); the donor and acceptor splice sites, which are involved in the processing (splicing) of mRNA; the termination codon, which interrupts translation; and the polyadenylation signal, which is crucial for the addition of a poly (A) tail to the mRNA. The importance of these critical sequences is underscored by the fact that nucleotide substitutions that either alter them or create new similar consensus sequences in a globin gene cause abnormal mRNA processing and constitute the molecular basis for most types of thalassemia. Detailed information on positions, genotypes, and phenotypes for the known globin gene variants are available at the websites listed at the end of this chapter. Essentially the process of globin gene expression consists of the following steps: Transcription of DNA into a primary mRNA transcript; processing of the primary mRNA, involving modifications at both its 5′ (capping) and 3′ (polyadenylation) ends together with removal of the introns and joining of the exons (splicing) to produce mature mRNA, the final template for protein synthesis; and translation of mRNA in the globin protein. Transcription and RNA processing occur in the nucleus, while translation occurs in the cytoplasm [for review, see (15, 60)]. Thalassemia syndromes result from a large series of molecular defects, which alter the expression of one or more globin genes.
of the β-globin gene.56, 57, 58, 59 Like other genes, globin genes possess a series of motifs critical for their expression: the CAP site, which indicates the start of transcription; the AGT initiation codon, which is the signal for starting translation in messenger RNA (mRNA); the donor and acceptor splice sites, which are involved in the processing (splicing) of mRNA; the termination codon, which interrupts translation; and the polyadenylation signal, which is crucial for the addition of a poly (A) tail to the mRNA. The importance of these critical sequences is underscored by the fact that nucleotide substitutions that either alter them or create new similar consensus sequences in a globin gene cause abnormal mRNA processing and constitute the molecular basis for most types of thalassemia. Detailed information on positions, genotypes, and phenotypes for the known globin gene variants are available at the websites listed at the end of this chapter. Essentially the process of globin gene expression consists of the following steps: Transcription of DNA into a primary mRNA transcript; processing of the primary mRNA, involving modifications at both its 5′ (capping) and 3′ (polyadenylation) ends together with removal of the introns and joining of the exons (splicing) to produce mature mRNA, the final template for protein synthesis; and translation of mRNA in the globin protein. Transcription and RNA processing occur in the nucleus, while translation occurs in the cytoplasm [for review, see (15, 60)]. Thalassemia syndromes result from a large series of molecular defects, which alter the expression of one or more globin genes.
 FIGURE 34.1. α– and β-globin gene cluster and hemoglobins (Hbs) produced during development. LCR, locus control region. |
 FIGURE 34.2. Current hemoglobin switching model. Bcl11A, B cell lymphoma 11; KLF, Krueppel-like factor; LCR, locus control region. (Reprinted with permission of Cao A, Moi P, Galanello R. Recent advances in β-thalassemias. Pediatr Rep 2011;3:e17.) |
α-Thalassemia
The α-globin genes are duplicated and located in the telomeric region of chromosome 16 (16p13.3) in a cluster containing an embryonic α-like gene (ζ2) and three pseudogenes (pseudo ζ1, pseudo α1, pseudo α2) (Fig. 34.1).61 A gene (θ) with unknown function, but whose mRNA can be found through all stages of development, is part of the α cluster. Several regions of the cluster contain tandem arrays of short GC-rich sequences (minisatellites), identified as hypervariable regions, and many Alu family repeats.62, 63 The α cluster is surrounded by widely expressed genes (MPG, C16 orf 35 and Luc 7L). Upstream of the α cluster there are four highly conserved noncoding sequences or multispecies conserved sequences (MCS), called MCS-R1-R4, that are involved in the regulation of the α-like globin genes.64 When the multipotent hemopoietic progenitors committed to the erythroid lineage start their differentiation to mature erythrocytes, several specific erythroid transcription factors, including GATA-1, GATA-2, SCL NF-E2, and cofactors, such as FOG, pCAF, and p300, bind to the MCS-R elements and the α-like globin gene promoters, causing extended modifications associated with chromatin activation. Then, RNA polymerase II is engaged both at the upstream regulatory regions and at the globin gene promoters beginning transcription in erythroid progenitors.64 The importance of the MCS as regulatory elements is suggested by the presence of rare deletions of this region that produce α-thalassemia, although both
α genes on each chromosome are intact (see below). The human α-globin gene cluster of apparently normal individuals contains a series of DNA sequence variations (e.g., single nucleotide polymorphism [SNP], variations in the number of tandem repeats [VNTRs]; and copy number variants [CNVs], which have been of considerable value in the analysis of evolutionary aspects of the gene cluster, in defining the origin of many of the α-thalassemia mutations, and in identifying functionally important areas of the cluster.)65, 66 The α complex is arranged in the order in which it is expressed during development: 5′ζ2 … α2–α1. There is a very high homology between α2 and α1 genes; they differ only in the IVS-2 (two base substitutions and a 7-bp insertion/deletion) and in the 3′ noncoding region (18 base substitutions and a single-base deletion in the 3′ untranslated region).67, 68 This remarkable homology has been maintained during evolution through repeated rounds of gene conversion.67, 68, 69 The embryonic ζ gene shows only 58% homology with the α genes in the coding region. The level of transcription of the two α genes differs: the α2 gene expresses two to three times more α-globin than α1.70, 71 This would imply that the globin structural variants of the α2 gene should represent about 35% of the total hemoglobin, while the α1 globin mutants should represent about 15%. However, contrasting results have been reported on this point. Shakin and Liebhaber have reported identical translation profiles of α2– and α1-mRNA, and higher percentages of α2-globin variants (24% to 40% as compared to 11% to 23% for the α1-globin variants).72 Molchanova et al. confirmed the average ratio of 2.6:1, observed for α2– and α1-mRNA, but reported an average percentage of the abnormal hemoglobin in heterozygotes with α2 mutations (23.5%) to be only slightly higher than that in heterozygotes with α1 mutations (19.7%), suggesting a less efficient translation of α2-mRNA.73 It should be pointed out that, besides the rate of transcription and efficiency of translation, other factors, such as the stability of the variant, the affinity of the variant for β-chains, and the number of active α genes, may influence the final level of the abnormal hemoglobin. The issue of different expression of the two α genes is important not only for the α-globin structural variants, but also for the pathophysiology of the deletional and nondeletional forms of α-thalassemia. Normal individuals have usually four α-globin genes, but as a result of unequal genetic exchange, some may have five or six α genes while still being phenotypically normal.74, 75, 76 Multiple arrangements with three to six ζ-like embryonic genes have also been reported.77,78
α genes on each chromosome are intact (see below). The human α-globin gene cluster of apparently normal individuals contains a series of DNA sequence variations (e.g., single nucleotide polymorphism [SNP], variations in the number of tandem repeats [VNTRs]; and copy number variants [CNVs], which have been of considerable value in the analysis of evolutionary aspects of the gene cluster, in defining the origin of many of the α-thalassemia mutations, and in identifying functionally important areas of the cluster.)65, 66 The α complex is arranged in the order in which it is expressed during development: 5′ζ2 … α2–α1. There is a very high homology between α2 and α1 genes; they differ only in the IVS-2 (two base substitutions and a 7-bp insertion/deletion) and in the 3′ noncoding region (18 base substitutions and a single-base deletion in the 3′ untranslated region).67, 68 This remarkable homology has been maintained during evolution through repeated rounds of gene conversion.67, 68, 69 The embryonic ζ gene shows only 58% homology with the α genes in the coding region. The level of transcription of the two α genes differs: the α2 gene expresses two to three times more α-globin than α1.70, 71 This would imply that the globin structural variants of the α2 gene should represent about 35% of the total hemoglobin, while the α1 globin mutants should represent about 15%. However, contrasting results have been reported on this point. Shakin and Liebhaber have reported identical translation profiles of α2– and α1-mRNA, and higher percentages of α2-globin variants (24% to 40% as compared to 11% to 23% for the α1-globin variants).72 Molchanova et al. confirmed the average ratio of 2.6:1, observed for α2– and α1-mRNA, but reported an average percentage of the abnormal hemoglobin in heterozygotes with α2 mutations (23.5%) to be only slightly higher than that in heterozygotes with α1 mutations (19.7%), suggesting a less efficient translation of α2-mRNA.73 It should be pointed out that, besides the rate of transcription and efficiency of translation, other factors, such as the stability of the variant, the affinity of the variant for β-chains, and the number of active α genes, may influence the final level of the abnormal hemoglobin. The issue of different expression of the two α genes is important not only for the α-globin structural variants, but also for the pathophysiology of the deletional and nondeletional forms of α-thalassemia. Normal individuals have usually four α-globin genes, but as a result of unequal genetic exchange, some may have five or six α genes while still being phenotypically normal.74, 75, 76 Multiple arrangements with three to six ζ-like embryonic genes have also been reported.77,78
The α-thalassemias are classified generally into the α° thalassemias, in which there is absence of α-chain production from the affected chromosome, and the α+ thalassemias, in which the production of the α-chains from the mutated chromosome is reduced.
Deletion α-Thalassemia
α-Thalassemia is caused most frequently by deletions of DNA that involve one or both α-globin genes. The α-globin genes are embedded within two highly homologous regions extending for about four kb, whose sequence homology has been maintained by gene conversion and unequal crossover events.61, 79 Three homologous subsegments (X, Y, and Z) separated by nonhomologous elements have been defined. Reciprocal recombination between Z boxes, which are 3.7 kb apart, and between X boxes, which are 4.2 kb apart, gives rise to chromosomes with only one α-gene. These α-thalassemia determinants, which are the most common, are referred to as –α 3.7 kb rightward deletion and –α 4.2 kb leftward deletion, respectively (Fig. 34.3).80 Based on the exact location within the Z box where the crossover took place, the –α 3.7 kb deletion is further subdivided into –α 3.7 I, –α 3.7 II, and –α 3.7 III.69 Besides the deletion α-thalassemia determinants, the nonreciprocal crossover produces chromosomes with three α-globin genes: ααα anti-3.7 and ααα anti-4.2.81, 82 More complex recombination events result in chromosomes with four or five α genes.73 In addition to the common –α 3.7 kb and –α 4.2 kb deletions, an increasing number of deletions that produce α+-thalassemia have been reported.83, 84 Most of the deletions are rare or highly region-specific. The result of a single α-globin gene deletion is a reduced production of α-chains from the affected chromosome (α+-thalassemia). Measurements of α-globin mRNA in patients with –α 4.2 determinants suggest that there is a compensatory increase in expression of the remaining α1 gene, while in the chromosome with –α 3.7 deletion, the remaining α gene is expressed roughly halfway between a normal α2 and α1 gene.85, 86 These differences in expression most likely are a consequence of changes in the rate of transcription, due to the new combinations of flanking sequences, to the modification in chromatin structure resulting from the deletion, or to variation in the interaction with the HS-40 (MCS-R2) regulatory element.87 Deletions that remove all or part of the α-globin gene cluster, including both α genes (entirely or in part) and sometimes the embryonic ζ2 gene, result in α0-thalassemia. The extent of the deletions, completely removing both α-globin genes, is from 100 kb to over 250 kb and sometimes other flanking genes, such as a DNA repair enzyme, a protein disulphide isomerase, and several anonymous housekeeping genes, are removed (Fig. 34.3).66, 84 However, interestingly, in subjects with these large deletions, the only phenotypical manifestation is α-thalassemia. Several molecular mechanisms (illegitimate recombination, reciprocal translocation, truncation of chromosome 16) have been described as responsible for these large deletions.63 The associated phenotype is the α0-thalassemia phenotype. At present approximately 50 deletions completely or partially deleting both α-globin genes, therefore resulting in α0 thalassemia, have been reported.64, 66 A series of naturally occurring human deletions that remove MCS regulatory elements have been identified.88, 89, 90 Despite the presence of intact α-globin genes of carriers, these deletions have the α° thalassemia phenotype. A shown by human deletions and by studies of transgenic mice, among the four regulatory elements (MCS-R1 to R4), the most relevant for α-globin gene expression is MCS-R2, located 40 kb upstream from the ζ globin gene.91 An interesting patient with a severe hemoglobin H disease phenotype and all four α intact genes, but lacking MCS-R2 in both chromosomes and also MCS-R1 and R3 in one chromosome, has been recently described.92 This report proves that the complete loss of the major MCS-R2 regulatory element severely downregulates α-globin gene expression but is not associated with the complete absence of α-chain production. A novel deletion involving the α1 and θ gene that inactivates the intact α2 gene has been reported.93 Subsequent studies have shown that the deletion juxtaposes to the structural normal α2 globin gene a downstream widely expressed gene (Luc 7L).94 Transcription of antisense in RNA from Luc 7L through the α2 gene mediates methylation of the associated CpG island with silencing of α2 globin gene expression. These findings identify a new mechanism underlying human genetic disease.
Nondeletion α-Thalassemia
Single nucleotide mutations or oligonucleotide deletions/insertions in regions critical for α-globin gene expression produce α-thalassemia. Nondeletional α+-thalassemia are relatively common, except the Constant Spring mutation, which is quite frequent in Southeast Asia. Several molecular mechanisms (mutations affecting RNA splicing, the poly [A] addition signal, the initiation of mRNA translation, chain termination mutations, in-frame deletions, frameshift mutations) and at least 72 well-defined types of nondeletion α-thalassemia have been described.84 The majority (50) of nondeletional mutants occur in the α2 gene and, as expected, have a more severe effect on α-globin gene expression, while 17 have been reported in the α1 gene, and 5 on a –α chromosome. Hb Constant Spring (α 142 TAA→CAA, StopSGln) is the most common of the nine potential chain termination mutants, which
change the stop codon to one amino acid, allowing mRNA translation to continue to the next in phase stop codon located within the polyadenylation signal. The result of this class of mutation is the production of a very low amount (˜1%) of an α-chain variant elongated by 31 amino acids. It has been suggested that the reason for the reduced production of the elongated variants is the instability of the mRNA due to disruption of the 3′ untranslated region.95 Other extended α-chain variants are Hb Icaria (α 142 Lys), Koya Dora (α 142 Ser), Seal Rock (α 142 Glu), and Paksé (α 142 Tyr). Heterozygotes for α-globin elongated chains, besides the presence of a very small amount of the hemoglobin variant, have the phenotype of α-thalassemia. Mutations of α-globin genes, which result in the production of highly unstable globin variants such as Hb Quong Sze (α 125 Leu→Pro), Hb Heraklion (α 37 Pro→0) and Hb Agrinio (α 29 Leu→Pro), unable to assemble in stable tetramers and thus rapidly degraded, produce the phenotype of α-thalassemia.96 A regularly updated overview of these variants can be found at the Hb Var website. A novel mechanism for nondeletion α-thalassemia has been suggested to explain the α-thalassemia phenotype with intact α genes in some Melanesian patients.64 Among 283 single nucleotide polymorphisms (SNP) identified by resequencing of approximately 213 Kb of DNA containing the α cluster and the surrounding regions, one SNP creating a GATA-1 binding site, always linked with α-thalassemia phenotype, has been identified.97 This new GATA-1 site, which binds transcription factors in vivo and is activated in erythroid cells because it is located closer to MCS elements, competes most efficiently with the α-globin promoters, thereby causing α-thalassemia.
change the stop codon to one amino acid, allowing mRNA translation to continue to the next in phase stop codon located within the polyadenylation signal. The result of this class of mutation is the production of a very low amount (˜1%) of an α-chain variant elongated by 31 amino acids. It has been suggested that the reason for the reduced production of the elongated variants is the instability of the mRNA due to disruption of the 3′ untranslated region.95 Other extended α-chain variants are Hb Icaria (α 142 Lys), Koya Dora (α 142 Ser), Seal Rock (α 142 Glu), and Paksé (α 142 Tyr). Heterozygotes for α-globin elongated chains, besides the presence of a very small amount of the hemoglobin variant, have the phenotype of α-thalassemia. Mutations of α-globin genes, which result in the production of highly unstable globin variants such as Hb Quong Sze (α 125 Leu→Pro), Hb Heraklion (α 37 Pro→0) and Hb Agrinio (α 29 Leu→Pro), unable to assemble in stable tetramers and thus rapidly degraded, produce the phenotype of α-thalassemia.96 A regularly updated overview of these variants can be found at the Hb Var website. A novel mechanism for nondeletion α-thalassemia has been suggested to explain the α-thalassemia phenotype with intact α genes in some Melanesian patients.64 Among 283 single nucleotide polymorphisms (SNP) identified by resequencing of approximately 213 Kb of DNA containing the α cluster and the surrounding regions, one SNP creating a GATA-1 binding site, always linked with α-thalassemia phenotype, has been identified.97 This new GATA-1 site, which binds transcription factors in vivo and is activated in erythroid cells because it is located closer to MCS elements, competes most efficiently with the α-globin promoters, thereby causing α-thalassemia.
 FIGURE 34.3. Most common deletional α-thalassemia defects. |
β-Thalassemia
The β-globin gene is located in the short arm of chromosome 11 in a region containing also the δ gene, the embryonic ε gene, the fetal Gγ and Aγ genes, and the pseudogene β1 (Fig. 34.1).98 The five functional globin genes are arranged in the order of their developmental expression. The complete sequencing of the β-globin gene complex has shown interspersed repetitive sequences (microsatellite repeats of [CA]n, an [ATTTT]n repeat, AluI and KpnI families of repeat DNA sequences), which may play a role in the generation of the deletions of the β cluster. The region also contains many polymorphic base substitutions, which produce restriction fragment length polymorphisms (RFLPs), combined in a restricted number of haplotypes in linkage disequilibrium with β-thalassemia mutations.99 Haplotype analysis provides information relevant for population genetics of the hemoglobinopathies. A1 with the α-like globin genes, variations in the number of β cluster genes, mostly involving the γ genes (which may be present in one to five copies), have been reported.100, 101 Like the α genes, β-globin genes are
subject to a very complex regulatory mechanism, acting at the level of single genes as well as of the entire β cluster.
subject to a very complex regulatory mechanism, acting at the level of single genes as well as of the entire β cluster.
The appropriate expression of the different β-like globin genes in erythroid tissues during development depends on a major regulatory region highly conserved in mammals named the locus control region (LCR), located 5 to 25 kb upstream from the ε-globin gene.102 Five DNAase hypersensitive sites (HSs) have been described in this region and each HS contains one or more binding motifs for erythroid-specific transcriptional activator1 (GATA-1 and NF-E2) and for ubiquitous DNA-binding proteins.103 The importance of the LCR for the control of β-like globin gene expression has also been suggested by a series of naturally occurring deletions that totally or partially remove the HS sites and result in the inactivation of the intact downstream β-globin gene.104, 105 β-Thalassemia mutations result in either a complete absence of β-globin chains (β0-thalassemia) or in a largely variable reduction of β-globin output (β+-thalassemia). More than 200 different mutations producing β-thalassemia have been so far described; the large majority are point mutations in functionally important sequences of the β-globin gene; while, in contrast to α-thalassemia, gene deletion is a rare cause of β-thalassemia (Table 34.1). A complete updated list of β-thalassemia mutations has been published and is also available through the globin gene server websites (see the end of the chapter).
Nondeletion β-Thalassemia
Point mutations resulting in β-thalassemia are single nucleotide substitutions or oligonucleotide insertions/deletions that affect the β gene expression by a variety of mechanisms (Table 34.1).
Three main categories can be identified: (a) mutations altering β gene transcription (promoter and 5′ untranslated region mutants); (b) mutations affecting mRNA processing (splice junction and consensus sequence mutants, exon and intron cryptic site mutants, the polyadenylation site and other 3′ untranslated region mutants); and (c) mutations resulting in abnormal mRNA translation (nonsense, frameshift, and initiation codon mutants).
Transcription Mutations
Promoter Mutations
Several mutations have been described in or around the conserved motifs in the 5′ flanking sequence of β-globin genes (TATA box, proximal and distal CACCC box). They reduce binding of RNA polymerase, thereby reducing the rate of mRNA transcription to 20%-30% of normal. They result in a moderate decrease of β-globin chain output (β+-thalassemia) and hence in a mild phenotype. One mutation C→T at position -101 to the β-globin gene (distal CACCC box) is unusually mild and associated with a silent phenotype in carriers and in a very mild thalassemia intermedia clinical picture in genetic compounds with severe β-thalassemia mutations.106, 107 The promoter mutations -28 A→G and -29 A→G are relatively common in Chinese and black populations, while -87 C→G and the silent -101 C→T have been described in the Mediterranean population. The first mutation in the conserved CCAAT box at positions -76 to -72 of the β-globin gene (-73 A→T) has been described in a Chinese patient.108
5′ Untranslated Region Mutations
Several mutations (single-base substitution and minor deletions) have been reported in this 50-nucleotide region; all have mild effects on gene transcription. Heterozygotes have normal or borderline red cell indices and HbA2; and compound heterozygotes, with severe β-thalassemia alleles, usually have a mild phenotype. The only homozygous state for a mutation at the β-globin gene mRNA capsite (Cap +1 ASC) shows hematologic values of a thalassemia trait.109
TABLE 34.1 MUTATIONS CAUSING β-THALASSEMIA | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Mutations Affecting mRNA Processing
RNA processing essentially consists in the removal of the intervening sequences and in the splicing of the coding regions to produce functional mRNA. The precision of this process relies on critical sequences present at intron/exon boundaries: the invariant dinucleotides —GT—at the 5′ (donor) and—AG—at the 3′ (acceptor) splice junctions and the flanking sequences (consensus sequences) that are rather well conserved.110
Splice Junction and Consensus Sequence Mutants
Mutations of the invariants 5′—GT— and 3′—AG—dinucleotides completely abolish normal splicing and result in β0-thalassemia. Twenty-seven base substitutions or short deletions involving the invariant dinucleotides have been identified. Other cryptic
splice sites present elsewhere in precursor mRNA are used for alternative splicing, but the misspliced mRNA cannot be translated into functional β-globin.111, 112 The efficiency of normal splicing may be decreased by mutations within the consensus sequences immediately adjacent to the splice junctions. The reduction of β-globin production is quite variable and the resulting phenotypes range from mild to severe. For example, the mutations at position five of IVS-1 (G→C, G→T, G→A) produce a consistent reduction of β-globin synthesis and hence a severe β+-thalassemia phenotype; while the IVS-1-6 T→C mutation (Portuguese mutation), quite common in Mediterraneans, only mildly affects normal splicing and results in a mild thalassemia intermedia clinical picture.113 Even in the consensus sequence mutations, abnormal alternative splicing using neighboring cryptic sites may occur.112
splice sites present elsewhere in precursor mRNA are used for alternative splicing, but the misspliced mRNA cannot be translated into functional β-globin.111, 112 The efficiency of normal splicing may be decreased by mutations within the consensus sequences immediately adjacent to the splice junctions. The reduction of β-globin production is quite variable and the resulting phenotypes range from mild to severe. For example, the mutations at position five of IVS-1 (G→C, G→T, G→A) produce a consistent reduction of β-globin synthesis and hence a severe β+-thalassemia phenotype; while the IVS-1-6 T→C mutation (Portuguese mutation), quite common in Mediterraneans, only mildly affects normal splicing and results in a mild thalassemia intermedia clinical picture.113 Even in the consensus sequence mutations, abnormal alternative splicing using neighboring cryptic sites may occur.112
Cryptic Site Mutants in Introns and Exons
Along introns and exons there are sequences similar to those found at the intron/exon boundaries, which normally are not used for splicing (“cryptic” splice sites). A number of nucleotide substitutions involving these sequences transform a cryptic site into a legitimate one. This new splice signal competes with the normal consensus sequence for splicing and, in some cases, is used preferentially (up to 90% in the IVS-1-110 G→A substitution and almost 100% in the IVS-1-116 T→G substitution), resulting in a severe β+– or β0-thalassemia phenotype.114, 115 Two cryptic splice site mutations in IVS-1 and three in IVS-2 have been described. In the exons, three cryptic splice sites can be activated by nucleotide substitution: one at codon (cd) 10 (C→A), a second at codon 19 (A→G), and a third by mutations at codons 24 (T→A), 26 (G→A), 26 (A→C), or 27 (G→T). The nucleotide substitutions partially activate the cryptic splice sites, resulting in both normally and abnormally spliced β-mRNA. Mutations at codon 19, 26, and 27 result in the production of abnormal hemoglobins (cd 19, Hb Malay [Asn→Ser], cd 26 HbE [Glu→Lys], cd26 Hb Tripoli [Glu→Ala] cd 27 Hb Knossos [Ala→Ser]) and are associated with a mild or silent phenotype because of the preferential use of the normal splice sites.112, 116, 117, 118
Poly (A) and Other 3′UTR Mutants
Downstream of the mRNA terminal codon there is a highly conserved AAUAAA sequence, which represents a signal for cleavage and polyadenylation reaction, as a part of the RNA transcript processing. Since polyadenylation is important in determining the stability of mRNA, mutations at the AAUAAA sequence affect the efficiency of translation, resulting in β+-thalassemia of variable, but usually mild, severity. Seven nucleotide substitutions at different positions, two oligonucleotide deletions (of two and five bases), and one deletion of the total AATAAA sequence have been described. Other mutations in the 3′ untranslated region (+1480 C→G) also produce β+-silent thalassemia.
Mutations Affecting mRNA Translation
A very large group of mutations alter the different steps of mRNA translation. Three categories of mRNA translation mutations can be identified: initiation codon mutations, nonsense mutations, and frameshift mutations.
Initiation Codon Mutations
The initiator codon ATG, which encodes for methionine, is a critical signal for starting translation. Nine different point mutations of the initiation codon have been reported as a cause of β0-thalassemia.
Nonsense Mutations
Single nucleotide substitutions may change a codon for a given amino acid to one of the three possible chain termination codons: TAA, TAG, or TGA. The result is a premature interruption of mRNA translation, with absence of β-globin production (β0-thalassemia). A very low level of β-mRNA has been detected in erythroid cells affected by mutations in exons 1 and 2 as a consequence of rapid degradation of the mutant β-mRNA.119, 120 This process is referred to as nonsense-mediated decay and may be a mechanism to eliminate mRNAs encoding truncated polypeptides, with potential harmful effects for the erythroid cell.121, 122 Nonsense mutations in exon 3 are associated with β-mRNA levels comparable with normal levels. The protective process does not occur, and mutant β-mRNA is probably translated to produce the abnormal globin (see Hyperunstable Globins, below).123
The most common nonsense mutation in the Mediterranean population, particularly in Sardinians, where it accounts for more than 95% of the cases of β-thalassemia, is the C→T base substitution at codon 39, while the nonsense mutation at codon 17 A→T shows a high frequency in the Chinese and Thai populations.124, 125
Frameshift Mutations
Insertion or deletion of one or a few nucleotides (other than three or multiples of three) alters the reading frame of the encoded mRNA starting at the site of the mutation. The new reading frame usually results in a novel abnormal amino acid sequence and in a premature termination further downstream. The mutant globin chain is rapidly degraded and the final result is a β0-thalassemia. The frameshift resulting from a single-base deletion at codon 6 (-A) is relatively common in the Mediterranean population, while the -4 nucleotides deletion at codons 41 and 42 are particularly common in Chinese and Asian Indian populations.126 The position of the premature termination (in exon 1, 2, or 3) caused by the frameshift mutation affects the mutant mRNA level and processing as previously reported for nonsense mutations.
β-Globin Gene Deletions
Several deletions affecting only the β-globin gene and ranging in size from 290 bp to about 67 kb have been reported. Only one, the 619-bp deletion, removing the 3′ end of the β-globin gene, is relatively common in the Sind and Punjab populations of India and Pakistan.127 All the others are extremely rare and have in common the deletion of the promoter region and at least part of the β-globin gene. The phenotype is that of β0-thalassemia with unusually high levels of HbA2 and F in heterozygotes. This is probably the result of the removal of competition for the upstream LCR, thus allowing an increased interaction between the LCR and the γ and δ gene in cis, with a consequently more efficient expression of these genes.128 Other deletions causing β-thalassemia remove either the whole β-globin cluster or the LCR. Total deletions of the β cluster result in lack of any globin production and, hence, in (ε Gγ Aγ δβ)0-thalassemia. Deletions of the β-LCR leaving the β gene intact inactivate the β gene. To date a total of ten deletions removing the whole β-globin cluster and eight removing the upstream LCR have been reported [original references in (129)]. These deletions confirm, in vivo, the critical importance of the LCR for the control of expression of the β-globin genes.
β-Thalassemic Hemoglobinopathies
This group includes some structurally abnormal hemoglobins associated with a thalassemia phenotype. They can be classified according to the molecular mechanism in:
δβ hybrid genes,
activation of cryptic splice sites,
hyperunstable β-globins, and
unknown mechanism.
δβ Hybrid Genes
Unequal crossing over between the homologous δ– and β-globin genes results in the formation of hybrid δβ and βδ genes, referred to as Lepore and anti-Lepore genes, respectively. The Lepore hemoglobins contain the N-terminal amino acid sequence of the normal δ-chain and the C-terminal sequence of the normal β-chain, and depending on the point of transition from δ to β sequence, three different variants of Hb Lepore have been described: Boston or Washington (δ87/βIVS-2-8), Baltimore (δ68/β84), and Hollandia (δ22/β IVS-1-16).130, 131, 132, 133 The rate of production of the Lepore hemoglobins (about 10% in the carriers) likely depends on the structure of the hybrid gene, which has the promoter of the δ gene (this would explain the lower Hb Lepore amount as compared to normal HbA); and the IVS-2 of the β gene, which probably contains an enhancer (this would explain the higher level of Hb Lepore as compared with HbA2). Moreover, a relative instability of the Lepore mRNA may be responsible for the low level of synthesis. Nonhomologous crossing over between the β and δ genes also results in the production of a hybrid βδ gene in a chromosome also containing the normal β and δ genes. These anti-Lepore genes produce about 15% to 20% of the abnormal hemoglobin. Based on the position of the fusion point, several anti-Lepore hemoglobins have been identified (Hb Miyada, P Congo, P Nilotic, and Hb Lincoln Park, which has in addition a valine residue deleted at position 137), and carriers have normal hemoglobin levels and normal red cell indices.
A similar nonhomologous crossing over involving the Aγ– and β-globin gene produces an abnormal hybrid chain, which contains γ and β sequences (Hb Kenya). Restriction enzyme analysis in these patients shows a deletion of about 22.5 kb and the loss of sequences extending from exon 2 of the Aγ gene to exon 2 of the β gene.134
Activation of Cryptic Splice Sites
This group, including the HbE, Hb Malay, and Hb Knossos, has been previously described.
Hyperunstable Globins
A singular group of β-globin gene mutants are characterized by amino acid substitutions, additions, or deletions in the β-globin chain associated with a clinically detectable thalassemic phenotype in the heterozygous state. For this reason, these forms are also referred to as dominantly inherited β-thalassemia. The molecular lesions include 12 missense mutations, 9 small deletions or insertions of intact codons resulting in severe β-globin destabilization, 2 premature terminations, and 23 frameshift or aberrant splicing producing elongated or truncated β-globin chains. Most of these mutations are located in the exon 3.135
In contrast with the typical recessively inherited forms of β-thalassemia, which lead to a reduced synthesis of normal β-globin chains, this group of mutations results in the production of β-globin variants, which are extremely unstable. These hyperunstable globins fail to form functional tetramers and precipitate in the erythroid precursors, leading to ineffective erythropoiesis, which is exacerbated by the concomitant relative excess of α-chains. Most of the patients present the phenotype of thalassemia intermedia; a few patients have a thalassemia trait; and some may even have a severe anemia requiring red blood cell transfusions. Laboratory findings consist of varying degrees of hypochromic microcytic anemia, increased HbA2 and an imbalanced α– to β-globin synthesis ratio. In most of the cases the hemoglobin variant cannot be detected in the peripheral blood.
Unknown Mechanism
Adams et al. reported a patient with 8% of an abnormal hemoglobin (Hb Vicksburg β75 Leu→0) and the phenotype of thalassemia intermedia.136 The reason for the thalassemia intermedia phenotype associated with Hb Vicksburg has not yet been defined.
The original patient has been re-examined and, despite the use of the new technologies of DNA analysis, the predicted Hb Vicksburg deletion was not present.137 Moreover, even the Hb variant was not detected on two occasions, while HbA, absent at the beginning, has now been found. DNA analysis showed that the patient was a compound heterozygote for the -88 C→T β+ allele and IVS- 2-849 A→G mutation that causes β0-thalassemia.137, 138 It has been proposed that Hb Vicksburg arose as a stem cell mutation on the β+-thalassemia chromosome. The variable hemoglobin composition at different ages suggests that over time there were at least two clones of erythroid progenitors contributing to erythropoiesis.137, 138 A phenotype of mild heterozygous β-thalassemia with microcytosis and increased levels of HbA2 has been reported in patients with two hemoglobin variants: Hb North Shore (β134 Val→Glu) and Hb Woolwich (β132 Lys→Glu).138, 139 In both cases, a mild deficit of β-globin chain synthesis has been reported. DNA analysis of these patients has not been performed and the mechanism responsible for the thalassemic phenotype remains unknown.
δ-Thalassemia
Several mutations of the δ-globin gene, which result in reduced (δ+-thalassemia) or absent (δ0-thalassemia) production of δ-globin chains, have been described. These conditions do not have clinical relevance, but the coinheritance with β-thalassemia mutations may create problems in β-carrier identification, since the HbA2 may be normal or borderline. The classes of mutations are similar to those responsible for β-thalassemia. Some δ-thalassemia mutations have been described in cis to β-thalassemia. The δ+27 C→T, fairly common in the Mediterranean population, has been reported in cis to β+IVS-2-745 C→G, β039 C→T, and β+27 G→T (Hb Knossos).140, 141, 142 Also the Corfu deletion (-7.2 kb) has been reported isolated or associated with the β+IVS-1-5 G→A mutation.143, 144
δβ-Thalassemia
δβ-Thalassemia includes a group of disorders characterized by reduced or absent production of both δ– and β-globin chains and by a variable increase in γ-chain synthesis, which is only partially able to balance the δ– and β-chain deficiency. The most common molecular mechanism consists of deletions of variable extent of the β-like globin cluster, which involve the δ– and β-globin genes. Based on the presence of one (Gγ) or both (Gγ and Aγ) globin genes, and hence on the residual synthesis of only Gγ– or both Gγ– and Aγ-globin chains, two groups of δβ0-thalassemia have been identified: Gγ (Aγ δβ)0– and Gγ Aγ(δβ0)-thalassemia. In Table 34.2 the different varieties with the size of the deletion are summarized. Some have been described in a single family or a few families, while others, such as the Sicilian, the Spanish Gγ.
Aγ(δβ0), and the black Gγ (Aγ δβ)0, are more common. Homozygotes have been reported as well. For some deletions, the 3′ breakpoint has not been defined. The majority of the deletions that result in δβ-thalassemia are due to illegitimate recombination. Similar but more complex mechanisms have been invoked to explain other δβ0-thalassemias such as Macedonian/Turkish Gγ Aγ(δβ)0 thalassemia, which is characterized by a double deletion/inversion rearrangement.145, 146 The reasons for the increased expression of the γ genes in δβ0-thalassemia and for the differences between δβ0-thalassemia
and hereditary persistence of fetal hemoglobin (HPFH, see below) have not been defined. Juxtaposition to the globin genes of new sequences as a result of the deletion; removal of intergene sequences critical for control of γ-globin gene expression; and altered spatial relationships between the LCR and the genes of the β cluster (with changes in LCR/globin gene promoter interaction and competition) have been postulated to explain the upregulation of the γ-globin genes and the phenotypic differences between δβ-thalassemia and deletion HPFH. It is possible that a combination of the above mechanisms plays a role, and that a balance between regulatory sequences, with positive or negative effects on the γ gene expression, may finally determine the amount of HbF in the red cells. A recent study of three families with elevated HbF identified using comparative genomic hybridization, breakpoint DNA sequencing, and chromatin immunoprecipitation identified a 3.5 kb intergenic region near the 5′ end of the β-globin gene, which is necessary for γ-globin gene silencing.147 This region binds the fetal hemoglobin silencing factor BCL11A and its partners in the chromatin of adult erythroid cells. The Corfu δβ-thalassemia is characterized by a deletion of 7.2 kb, which removes the δ gene associated with the β-IVS-1-5 G→A mutation.148 Carriers for this mutation have the unusual hematologic phenotype of heterozygous β-thalassemia with normal levels of HbA2, while homozygotes have relatively high levels of HbF and a mild clinical phenotype.144 The 7.2-kb deletion has been reported isolated as a deletion form of δ-thalassemia not associated with increased HbF.144, 145 Two varieties of nondeletion δβ-thalassemia have also been described. One, relatively common in Sardinia, is of the δβ0 type and presents two mutations in cis: the common β039 C→T nonsense mutation, and a point mutation at position -196 in the Aγ gene promoter, which is responsible for the Italian/Chinese nondeletion Aγ HPFH (see below).149 The other, reported in two Chinese families and characterized by decreased expression of the β-globin gene and increased expression of both Gγ and Aγ globin genes (δβ+-thalassemia), showed no deletion in the β-globin cluster.150 In one of these families, the -29 A→G mutation in the promoter of the β gene (a mild β+ allele) and a non-polymorphic C→T substitution in the 3Aγ enhancer have been identified.151, 152
and hereditary persistence of fetal hemoglobin (HPFH, see below) have not been defined. Juxtaposition to the globin genes of new sequences as a result of the deletion; removal of intergene sequences critical for control of γ-globin gene expression; and altered spatial relationships between the LCR and the genes of the β cluster (with changes in LCR/globin gene promoter interaction and competition) have been postulated to explain the upregulation of the γ-globin genes and the phenotypic differences between δβ-thalassemia and deletion HPFH. It is possible that a combination of the above mechanisms plays a role, and that a balance between regulatory sequences, with positive or negative effects on the γ gene expression, may finally determine the amount of HbF in the red cells. A recent study of three families with elevated HbF identified using comparative genomic hybridization, breakpoint DNA sequencing, and chromatin immunoprecipitation identified a 3.5 kb intergenic region near the 5′ end of the β-globin gene, which is necessary for γ-globin gene silencing.147 This region binds the fetal hemoglobin silencing factor BCL11A and its partners in the chromatin of adult erythroid cells. The Corfu δβ-thalassemia is characterized by a deletion of 7.2 kb, which removes the δ gene associated with the β-IVS-1-5 G→A mutation.148 Carriers for this mutation have the unusual hematologic phenotype of heterozygous β-thalassemia with normal levels of HbA2, while homozygotes have relatively high levels of HbF and a mild clinical phenotype.144 The 7.2-kb deletion has been reported isolated as a deletion form of δ-thalassemia not associated with increased HbF.144, 145 Two varieties of nondeletion δβ-thalassemia have also been described. One, relatively common in Sardinia, is of the δβ0 type and presents two mutations in cis: the common β039 C→T nonsense mutation, and a point mutation at position -196 in the Aγ gene promoter, which is responsible for the Italian/Chinese nondeletion Aγ HPFH (see below).149 The other, reported in two Chinese families and characterized by decreased expression of the β-globin gene and increased expression of both Gγ and Aγ globin genes (δβ+-thalassemia), showed no deletion in the β-globin cluster.150 In one of these families, the -29 A→G mutation in the promoter of the β gene (a mild β+ allele) and a non-polymorphic C→T substitution in the 3Aγ enhancer have been identified.151, 152
TABLE 34.2 MUTATIONS RESPONSIBLE FOR DELETIONAL AND NONDELETIONAL δβ-THALASSEMIA | ||||||||||||||||||||||||||||||||||||||||||||||||||||
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Hereditary Persistence of Fetal Hemoglobin
HPFH is characterized by the presence of increased levels of HbF in adult life in the absence of relevant hematologic abnormalities. The amount of HbF is quite variable, ranging in the carriers from 2.0% to 30%, and this variability reflects a marked molecular heterogeneity. Both deletion and nondeletion defects have been identified. The deletions resulting in HPFH, listed in Table 34.3, extend from 13 kb (HPFH-5 or Sicilian HPFH) to about 106 kb (HPFH-1 or black HPFH).153, 154 They remove δ– and β-globin genes, but spare both Gγ and Aγ genes. As in δβ0-thalassemia, the most common mechanism producing deletions is an illegitimate recombination followed by unequal crossing over. Nondeletion HPFH usually is the result of mutations in the promoter regions of Gγ and Aγ genes (Table 34.3). Most of these mutations are single nucleotide substitutions in or very close to the conserved sequences that bind various regulatory transcription factors. As a consequence, there are changes in the binding of repressor or activator proteins that may modify the balance of the competition between the promoter and the LCR, ultimately resulting in increased HbF synthesis in adult life.129 In some families, mostly with interacting β-thalassemia or sickle cell anemia, it has been shown that HPFH may segregate unlinked to the β-globin cluster.155, 156, 157 Several patterns of inheritance have been identified: autosomal or X-linked dominant and autosomal recessive. The locus for the X-linked form seems to reside at Xp22.2-22.3.158 Craig et al., by using polymorphic markers covering the whole genome to study a single very large family, localized a putative locus for HPFH at 6q22.3-q24.157 Candidate-gene association studies and genome-wide association studies allowed the identification of common single nucleotide polymorphism (SNPs) in the HBS1L-MYB intergenic region on chromosome 6p23, and in the IVS2 of BCL11A gene in chromosome 2p16.1, associated with increased HbF levels in healthy subjects, in β-thalassemia, and in sickle cell anemia.45, 159, 160, 161
Unusual Causes of β-Thalassemia
Insertion of a transposable element into IVS2 of the β-globin gene, resulting in the expression of approximately 15% of normal β-globin mRNA, has been reported with the phenotype of β-thalassemia.165 Mutations in the general transcription factor TFIIH, involved in basal transcription and DNA repair, cause trichothiodystrophy and are frequently associated with the
phenotype of the β-thalassemia trait.166 Some mutations in the erythroid transcription factor GATA have been reported as a cause of β-thalassemia associated with thrombocytopenia.167, 168 Large somatic deletions at chromosome 11 p15.5, including the β-globin cluster and leading to thalassemia intermedia, have been reported in heterozygous β-thalassemia patients.169, 170 The deletion in a subpopulation of erythroid cells resulted in a somatic mosaic with 10% to 20% of erythroid cells heterozygous with one normal copy of the β-globin gene, and the rest homozygous without any normal β-globin gene.
phenotype of the β-thalassemia trait.166 Some mutations in the erythroid transcription factor GATA have been reported as a cause of β-thalassemia associated with thrombocytopenia.167, 168 Large somatic deletions at chromosome 11 p15.5, including the β-globin cluster and leading to thalassemia intermedia, have been reported in heterozygous β-thalassemia patients.169, 170 The deletion in a subpopulation of erythroid cells resulted in a somatic mosaic with 10% to 20% of erythroid cells heterozygous with one normal copy of the β-globin gene, and the rest homozygous without any normal β-globin gene.
TABLE 34.3 MUTATIONS RESPONSIBLE FOR HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH) | ||||||||||||||||||||||||||||||||||||||||||||||||
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PATHOPHYSIOLOGY
Pathophysiology of the thalassemia syndromes is extremely complex and encompasses intracellular processes as well as their consequences for the organism as a whole. The complete comprehension of the pathophysiology of the thalassemias is essential for the understanding of the differences in clinical expression between α– and β-thalassemia, the high variability of the clinical phenotypes, and potential therapeutic treatment.
β-Thalassemia
The basic defect in β-thalassemia is a reduced or absent production of β-globin chains with relative excess of α-chains. The direct consequences are a net decrease of hemoglobin production and an imbalance of the globin chain synthesis. The former is more evident in carriers, leading to a reduction of mean cell hemoglobin (MCH) and mean cell volume (MCV), and has a minor clinical significance. The latter has dramatic effects on the red cell precursors, ultimately resulting in their extensive premature destruction in the bone marrow and in the extramedullary sites. This process is referred to as ineffective erythropoiesis and is the hallmark of β-thalassemia. Using ferrokinetic analysis, it has been shown that in β-thalassemia patients only 15% of 59Fe was incorporated in circulating erythrocytes, indicating that ineffective erythropoiesis could account for as much as 60% to 75% of total erythropoiesis.171, 172 Hemolysis of the erythrocytes containing inclusions that reach peripheral blood is a minor cause of anemia, particularly in thalassemia major (Fig. 34.4).
The excess α-chains may, in minor amounts, combine with residual β– (in β+-thalassemia) and γ-chains (whose synthesis persists usually in small quantity after birth), undergo proteolysis, or in large part become associated with the erythroid precursors and red cell membrane, with deleterious effects on erythroid maturation and survival. Therefore, the main determinant of the clinical severity is the extent of the relative excess of α-chains in red cell precursors and hence the degree of α/non-α imbalance. In 1966, Fessas et al. described the presence of inclusion bodies in erythroblasts of thalassemic patients, suggesting that they were precipitated by α-chains.174 The composition of inclusion bodies in β-thalassemia, completely consisting of precipitated α-chains, has been confirmed by immunoelectron microscopy.175 Oxidation of excess α-chains results in the formation of hemichromes, whose basic structure consists of the covalent binding of distal histidine E7 to the sixth coordination site of the heme iron. Irreversible hemichromes and denatured α-chains precipitate as inclusion bodies early during differentiation and throughout erythroid maturation.176α-Chain precipitation in the red cell membrane causes structural and functional alterations with changes in deformability, stability, and red cell hydration.174, 177 Isolated red cell membranes from β-thalassemia intermedia, particularly from splenectomized patients, are rigid and unstable. Protein 4.1, a major component of the cytoskeleton, undergoes partial oxidation in β-thalassemia, resulting in its defective capability to mediate the formation of the spectrin-protein 4.1-actin complex, which is critical to maintain cytoskeleton stability.178 In vitro experiments, using purified α-chains released within normal red blood cells, support the role of aggregated α-chains in causing red cell membrane rigidity.179 A further consequence of the membrane-bound hemichromes is their association with the cytoplasmic domain
of protein band 3, creating a neoantigen, which is subjected to opsonization with autologous immunoglobulin G (IgG) and complement and immune removal of the cell by macrophages.180, 181 Red blood cells in β-thalassemia lose K+, store Ca2+, and are dehydrated, resulting in altered deformability.174 Besides oxidation, free α-chains are subjected to degradation, resulting in the formation of denatured α-globin protein, heme, and free iron. These degradation products play a role in damaging erythroid precursors and red cell membranes.
of protein band 3, creating a neoantigen, which is subjected to opsonization with autologous immunoglobulin G (IgG) and complement and immune removal of the cell by macrophages.180, 181 Red blood cells in β-thalassemia lose K+, store Ca2+, and are dehydrated, resulting in altered deformability.174 Besides oxidation, free α-chains are subjected to degradation, resulting in the formation of denatured α-globin protein, heme, and free iron. These degradation products play a role in damaging erythroid precursors and red cell membranes.
 FIGURE 34.4. Pathophysiology of β-thalassemia. Ig, immunoglobulin. |
Free iron, via the Fenton reaction, generates reactive oxygen species, which cause lipid and protein peroxidation with consequent damage to red cell membranes and intracellular organelles.182 High levels of iron, closely associated with denatured hemoglobin, have been found in the membrane of β-thalassemic red cells.183 In vitro and in vivo experiments (in humans and animals) have shown that the oral iron chelator deferiprone, which may enter the cells, is able to remove free iron from thalassemic red cells, resulting in an improvement of red cell survival.184, 185 These results confirm the role of free iron in damaging the red cell membrane structure. Also, heme, and its oxidized form hemin, produce oxidative damage to the different components of the red cell membrane with consequent structural and functional alterations.177, 186 Recently a new protein relevant for α-globin stabilization has been discovered and its function deeply analyzed.187 This protein, named α-hemoglobin-stabilizing protein (AHSP), is an abundant erythroid protein that specifically binds free α-globin chains, stabilizes their structure, and limits their ability to participate in chemical reactions that generate reactive oxygen species.188
All the above-reported alterations of erythroid precursors cause their extensive premature death by apoptosis in the bone marrow, significantly contributing to ineffective erythropoiesis.189 Of the two major pathways of apoptosis, the mitochondrial pathway and the cell surface death domain pathway, the death domain pathway seems to be mainly involved.190 Both FAS and FAS ligand appear to be upregulated at all stages of β-thalassemic erythroid progenitors. Procaspase 8, one of the initiator caspases, whose activation is a hallmark of the death receptor domain pathway, is also activated.190 In turn, caspases inactivate GATA-1, a transcription factor necessary for erythroid growth maturation, thereby suppressing and limiting the thalassemic erythropoiesis.191 Other recent studies in mouse models of β-thalassemia have shown that increased or excessive activation of the Jak2/STAT5 pathways promotes unnecessary disproportionate proliferation of erythroid progenitors, exacerbating ineffective erythropoiesis.192
The ineffective erythropoiesis and anemia have several consequences in the organism as a whole, producing the clinical picture of the disease. The first response to anemia is an increased production of erythropoietin, causing a marked erythroid hyperplasia, which may range between 10 and 30 times normal.172 Anemia may produce cardiac enlargement and sometimes severe cardiac failure. Erythroid expansion produces skeletal deformities, osteoporosis, and occasionally extramedullary masses, and contributes to splenomegaly. Untreated or undertreated thalassemia major patients have retarded growth as a result of anemia and the excessive metabolic burden imposed by erythroid expansion. Environmental factors, such as poor nutrition and infections, may contribute to growth failure. The high vascularization of expanded marrow results in an increase in plasma volume, which, associated with splenomegaly, aggravates the anemia.
Ineffective erythropoiesis is associated with increased iron absorption, whose mechanism has been recently clarified.193. Expression of growth differentiation factor-15 (GDF-15), a TGF-β superfamily member, usually associated with cellular stress or apoptosis, has been found at particularly high levels in the serum of patients with thalassemia and other conditions associated with ineffective erythropoiesis. Elevated levels of GDF-15 contribute to the suppression of hepcidin, a small peptide produced in the liver, considered the major regulator of iron.194 Low levels of hepcidin result in increased intestinal iron absorption and most of that excess iron is stored in the liver.195 A second novel erythroid regulator of hepcidin expression, i.e., twisted gastrulation factor 1 (TWSG1), has been recently identified in thalassemic and human cells.196 In transfusion-dependent patients, iron overload is largely increased by repeated blood transfusions (see Complications of Transfusions). Iron overload damages several organs (myocardium, liver, endocrine glands), leading to severe complications. Removal of the abnormal red blood cells by the reticuloendothelial elements of the spleen results in splenomegaly and hypersplenism, which, when severe, may exacerbate anemia and cause thrombocytopenia and leukopenia.
A further consequence of the red blood cell membrane damage is the loss of the normal asymmetric distribution and increased surface exposure of the procoagulant, negatively charged phospholipids phosphatidylserine and phosphatidylethanolamine. Perturbation of the cell membrane causes thalassemic RBCs to become rigid and deformed, increasing their cohesiveness and tendency to aggregate.197 The anionic phospholipids increase thrombin generation, which leads to activation of platelets and endothelial cells.198, 199, 200 Further evidence of the chronic platelet activation has been well documented in patients with thalassemia, as evidenced by increased platelet aggregation and expression of CD62P (P-selectin) and CD 63, shorter platelet survival, and elevated levels of urinary metabolites of prostacyclin (PGI2) and thromboxane A2 (TXA2), two markers of hemostasis.197, 201, 202 Moreover, thrombocytosis and increased platelet aggregation associated with splenectomy contribute to the higher risk of thromboembolic events. Decreased levels of naturally occurring anticoagulants, such as protein C and protein S, and elevated plasma levels of the thrombin-antithrombin III complex, have been reported.203, 204 It has also been shown that adherence of thalassemic red cells to endothelial cells is markedly enhanced as compared to that of normal red cells, and elevated levels of endothelial adhesion proteins (intracellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, von Willebrand factor) in the serum and plasma of thalassemic patients have been described.205, 206 Activated monocytes and granulocytes, found in patients with thalassemia, could contribute to the endothelial damage and the hypercoagulable state.207, 208
α-Thalassemia
As in the case of β-thalassemia, the primary defect of α-thalassemia is the imbalance of globin biosynthesis, with an excess of β-and/or γ-globin chains. Unlike α-chains, which are highly unstable and unable to form soluble tetramers, excess γ-chains in fetal life and β-chains in extrauterine life associate to form relatively soluble γ4 tetramers (Hb Bart) and β4 tetramers (HbH), respectively. These excess non-α-chains damage mostly mature red cells and to a lesser extent erythroid precursors, leading mainly to hemolysis and minimally to ineffective erythropoiesis. The different characteristics of the excess chains in α-thalassemia are of great importance in determining its pathophysiology; but in addition, the functional properties of HbH and Hb Bart and the number of α genes contribute to the difference in severity of α-thalassemia as compared to β-thalassemia syndromes. Red blood cells in α-thalassemia are rigid, as in β-thalassemia, but unlike β-thalassemia, they are hyperhydrated and have red cell membranes that are hyperstable. The reason for hyperhydration has not been clearly defined, but is likely a consequence of the effect of β-chain excess on the KCl cotransporter system. Membrane skeletal-bound β-globins become partially oxidized, with consequent membrane damage. In vitro studies have shown that entrapment of β-chains in normal red cells does not result in any significant change in membrane protein function or thiol concentrations, but rather produces changes in red cell deformability,
as reported in vivo in patients with HbH disease.209, 210 Interaction of excess β-globin with the cytoplasmic domain of protein band 3 is abnormal in erythrocytes of patients with HbH disease, since β4 tetramers tend to adhere tightly to protein band 3.211 β-Globin tetramers precipitate as the red cell becomes old, forming inclusions. These inclusions can be induced in vitro by vital stains, such as brilliant cresyl blue or new methylene blue, and are more common in splenectomized patients.212 Studies using monoclonal antibodies have shown that red cell inclusions in HbH disease are composed of β-globin. Membrane-bound inclusion bodies perturb the flow velocity during transit through the spleen capillaries, ultimately resulting in mechanical trapping and macrophagic phagocytosis.213 Red cell hemolysis is a significant pathophysiologic mechanism of HbH disease, but ineffective erythropoiesis is a component, even if moderate. This has been suggested by morphologic and ferrokinetic studies and by the analysis of plasma levels of the transferrin receptor.214, 215 Excess β-chains accumulate and precipitate not only in old red cells, but also in marrow erythroid precursors, where they may cause some intramedullary cell death. β4 inclusion bodies alter the normal membrane phospholipid bilayer, exposing phosphatidylserine, which represents a signal for the development of apoptosis and red cell removal by the macrophages in the spleen and other reticuloendothelial organs.216, 217 Programmed cell death has been found moderately increased in patients with HbH disease. Red cell membrane deformability and stability is even more affected in patients with HbH/Hbcs.218 As compared with patients with HbH disease, HbH/Hbcs patients have a higher amount of HbH and a higher percentage of erythrocytes with inclusion bodies and with translocated phosphatidylserine.213, 217, 219 All these characteristics may account for the increased hematologic severity of HbH/Hbcs disease. γ-Globin tetramers (Hb Bart) are much less prone than β-globin tetramers to precipitate and form inclusions.220 Besides the characteristics of excess β– and γ-globin chains discussed above, other functionally abnormal properties are important in determining the pathophysiology of α-thalassemia. HbH and, even more, Hb Bart, have a very high oxygen affinity and show no heme/heme interaction or Bohr effect, hence severely reducing their oxygen-carrying capacity.221, 222 Some α-globin chain variants barely symptomatic in the heterozygous state, are either unstable because of folding defects and/or defective in binding to α-hemoglobin-stabilizing protein (AHSP) and are associated with the α-thalassemia phenotype.96 When the patients are homozygous for such variants or compound heterozygotes with another α thalassemic defect, they present the phenotype of HbH disease or chronic hemolytic anemia or even hydrops fetalis syndrome.
as reported in vivo in patients with HbH disease.209, 210 Interaction of excess β-globin with the cytoplasmic domain of protein band 3 is abnormal in erythrocytes of patients with HbH disease, since β4 tetramers tend to adhere tightly to protein band 3.211 β-Globin tetramers precipitate as the red cell becomes old, forming inclusions. These inclusions can be induced in vitro by vital stains, such as brilliant cresyl blue or new methylene blue, and are more common in splenectomized patients.212 Studies using monoclonal antibodies have shown that red cell inclusions in HbH disease are composed of β-globin. Membrane-bound inclusion bodies perturb the flow velocity during transit through the spleen capillaries, ultimately resulting in mechanical trapping and macrophagic phagocytosis.213 Red cell hemolysis is a significant pathophysiologic mechanism of HbH disease, but ineffective erythropoiesis is a component, even if moderate. This has been suggested by morphologic and ferrokinetic studies and by the analysis of plasma levels of the transferrin receptor.214, 215 Excess β-chains accumulate and precipitate not only in old red cells, but also in marrow erythroid precursors, where they may cause some intramedullary cell death. β4 inclusion bodies alter the normal membrane phospholipid bilayer, exposing phosphatidylserine, which represents a signal for the development of apoptosis and red cell removal by the macrophages in the spleen and other reticuloendothelial organs.216, 217 Programmed cell death has been found moderately increased in patients with HbH disease. Red cell membrane deformability and stability is even more affected in patients with HbH/Hbcs.218 As compared with patients with HbH disease, HbH/Hbcs patients have a higher amount of HbH and a higher percentage of erythrocytes with inclusion bodies and with translocated phosphatidylserine.213, 217, 219 All these characteristics may account for the increased hematologic severity of HbH/Hbcs disease. γ-Globin tetramers (Hb Bart) are much less prone than β-globin tetramers to precipitate and form inclusions.220 Besides the characteristics of excess β– and γ-globin chains discussed above, other functionally abnormal properties are important in determining the pathophysiology of α-thalassemia. HbH and, even more, Hb Bart, have a very high oxygen affinity and show no heme/heme interaction or Bohr effect, hence severely reducing their oxygen-carrying capacity.221, 222 Some α-globin chain variants barely symptomatic in the heterozygous state, are either unstable because of folding defects and/or defective in binding to α-hemoglobin-stabilizing protein (AHSP) and are associated with the α-thalassemia phenotype.96 When the patients are homozygous for such variants or compound heterozygotes with another α thalassemic defect, they present the phenotype of HbH disease or chronic hemolytic anemia or even hydrops fetalis syndrome.
GENOTYPE-PHENOTYPE CORRELATION IN THALASSEMIA
Progress in molecular biology and the wide availability of methods for DNA analysis have allowed for the definition of globin gene defects in thalassemia syndromes and for the understanding of the mechanisms of globin gene regulation and expression, and have partially elucidated the relationship between genotype and phenotype. This knowledge is helpful in clinical practice for planning the management of the patients, and in genetic counseling for the prediction of phenotype from genotype in couples at risk. In patients, the differentiation at presentation between thalassemia major and intermedia is essential to design the appropriate treatment. In fact, the prediction of a mild phenotype may avoid unnecessary transfusions and their complications, while the diagnosis of thalassemia major will allow an early start of the transfusion program, thus preventing hypersplenism and the red cell sensitization often associated with a delayed start of red cell administration.223 As reported above, in β-thalassemia the globin chain imbalance is the main determinant of clinical severity. Therefore, the presence of factors able to reduce the globin chain imbalance results in a milder form of thalassemia. These factors are the coinheritance of α-thalassemia or of genetic determinants that increase γ-chain production and the presence of silent or mild β-thalassemia alleles, associated with a high residual output of β-globin. Examples of these alleles are the silent -101 C→T and the mild IVS-1-6 T→C mutation in the Mediterranean population and the -29 A→G in Africans. Deletion and nondeletion HPFH mutations, associated with a high HbF level in carriers, when in genetic compounds with severe β-thalassemia alleles, result in mild thalassemia intermedia. A mild phenotype may also be determined by coinheritance of genetic determinants associated with γ-chain production, mapping outside the β-globin cluster. Several single nucleotide polymorphisms (SNPs) at the BCL11A gene on 2 p16.1 and HBS1L-MYB intergenic region on 6q23.3 have been associated with variable HbF levels in patients with thalassemia and sickle cell disease.45, 160, 224 The effect of α-thalassemia determinants in ameliorating the disease severity is less consistent, but the coinheritance of the deletion of two α-globin genes with homozygous β+-thalassemia, and sometimes even with β0-thalassemia, produces the clinical picture of thalassemia intermedia.15, 225 Variants at the three main quantitative trait loci (QTLs ) regulating HbF levels (i.e., BCL11A, HBS1L-MYB intergenic region, Xmn1 CG –γ gene) and α-thalassemia have been associated with the mild thalassemia intermedia phenotype and with a delayed need for transfusions in patients with homozygous β zero thalassemia.163, 226
The precise definition of the phenotype from the genotype is helpful also in genetic counseling, since it may avoid prenatal diagnosis in cases of expected very mild thalassemia intermedia in the fetus. Prenatal diagnosis in at-risk couples where the -101 C→T mutation is present should not be considered and the same applies to the coinheritance of the triple α-gene arrangement or of the HPFH mutations associated with high levels of HbF. The ameliorating effect that results from the presence of mild β-thalassemia alleles is less constant. The mild β-thalassemia allele IVS-1-6 T→C, common in the Mediterranean population, shows remarkable phenotypic diversity in some populations, such as the Jewish population.227 Despite the progress in better defining genetic determinants able to influence the clinical severity of β-thalassemia, phenotype prediction from genotype is not always accurate. However, the information obtained from extended genetic analysis may be used for planning appropriate management and for providing adequate genetic counseling, and may also reveal potential new targets for therapeutic intervention. As reported above, the wide range of phenotypic manifestations of thalassemia results from the heterogeneity of the primary mutation and from the coinheritance of other globin gene-associated determinants, which may ameliorate or worsen the disease severity.228 However, other known or unknown genetic determinants may modify the clinical expression of the thalassemia syndromes. Several secondary genetic modifiers have been identified in the recent years. The presence of (TA)7 polymorphism in the promoter region of the uridine diphosphoglucuronosyltransferase gene, which in the homozygous state is associated with Gilbert syndrome, is a risk factor for the development of cholelithiasis in thalassemia major and intermedia patients and in patients with HbE/β-thalassemia.229, 230 Other candidate genes for modification of the thalassemia phenotype are the apolipoprotein E ε4 allele, which seems to be a genetic risk factor for left ventricular failure in homozygous β-thalassemia.231 Less consistent data have been reported for genes involved in iron metabolism (C282Y and H63D HFE gene mutations), probably because their effect on iron overload is hidden as a result of treatment (e.g., secondary iron overload from red cell transfusion and iron chelation).232, 233
In α-thalassemia, the symptomatic form of HbH disease shows a wide phenotypic diversity. The phenotype varies depending on the number of α genes affected and on the type of mutation
present.234, 235 Studies that have correlated hematologic and clinical findings with α-globin genotypes indicate that HbH patients with nondeletion α-thalassemia defects have a more severe clinical expression (see below).236, 237, 238, 239
present.234, 235 Studies that have correlated hematologic and clinical findings with α-globin genotypes indicate that HbH patients with nondeletion α-thalassemia defects have a more severe clinical expression (see below).236, 237, 238, 239
Unlike with β-thalassemia, limited progress has been made in the search for genetic modifiers of HbH disease.238 One type of genetic modification is coinheritance of β-thalassemia mutations, also referred to as the HbH/β-thalassemia trait. Subjects with this genotype have severe hypochromia, microcytosis, and anemia, and do not present HbH at the electrophoresis.240
CLINICAL AND LABORATORY FEATURES
α-Thalassemia: Clinical Forms
Despite the large number of different α-thalassemia alleles (overall more than 100) only four hematologic and clinical conditions of increasing severity are recognized: silent carrier, α-thalassemia trait, HbH disease, and Hb Bart hydrops fetalis.241, 242
Silent Carrier
This condition results from the presence of a single α-globin gene defect associated with the 3.7 or 4.2 kb deletion (-α/αα) and from nondeletion defects. This genotype is characterized in the newborn period by a very mild increased percentage (1% to 2%) of Hb Bart, a tetramer of four γ-globin chains (γ4), which is produced when there is an excess of γ-chains in relation to α-chains. However, failure to demonstrate Hb Bart in cord blood does not exclude the silent carrier state.243, 244 Among black Americans, the incidence of the silent carrier state determined by gene mapping is about 27%, yet Hb Bart is detected in only 12% of cord samples. Similar trends have been found in Mediterranean and Saudi Arabian populations.245, 246
Adult individuals with three functional α genes may have a completely silent phenotype (normal red blood cell indices) or present a moderate thalassemia-like hematologic picture (reduced MCV and MCH and very mild anemia) with normal HbA2 and F.241, 242 Analysis of globin chain synthesis in vitro in peripheral blood reticulocytes displays a reduced α:β ratio in the range of 0.8 to 0.9. It has been shown that, at birth, children with the –α4.2 deletion, which removes the α2 gene, have a more severe phenotype than children with the –α3.7 deletion, which deletes most of the less productive α1 gene, resulting in a hybrid gene consisting of the 5′ part of the α2 gene linked to the 3′ part of the α1 gene.86, 246 However, with increasing age, the two genotypic forms become phenotypically indistinguishable, presumably because of upregulation of α-globin production by the α1-gene in subjects with the –α4.2 deletion.87, 247
α-Thalassemia Trait
This condition is characterized in the newborn by more markedly increased levels of Hb Bart (5% to 6%) and in the adult by thalassemia-like red cell indices, normal HbA2 and F, and a reduced α:β-globin chain synthesis ratio in the range of 0.7 to 0.8.241, 242 Subjects with two residual functional α genes, either in cis on the same chromosome (-/αα or α0-thalassemia carriers) or in trans in opposite chromosomes (-α/-α, homozygous α+-thalassemia), clearly show the α-thalassemia carrier state. Carriers of nondeletion defects have quite variable hematologic phenotypes ranging from the α-thalassemia trait to the silent carrier state (see above). Double heterozygotes for –α/and nondeletion α-thalassemia (-α/[αα]T) and homozygotes for nondeletion defects ([aa]T/[aa]T) have the typical phenotype of the α-thalassemia carrier state. However, homozygotes for some nondeletional forms of α-thalassemia may have a mild HbH disease.248 It should be pointed out that homozygotes for the Hb Constant Spring mutation, the most common nondeletion defect in the Asian population, have a clinical syndrome that is similar to HbH disease (see below).249 The α-thalassemia carrier state should be differentiated from iron deficiency and from δ– and β-thalassemia interaction (see carrier detection). This differentiation has important practical consequences.
Hemoglobin H Disease
HbH disease is common in Southeast Asia and relatively frequent in Mediterranean countries and parts of the Middle East, while occurring rarely in populations of African descent. This clinical condition results from the presence of only one functional α gene, usually as a consequence of the compound heterozygous state for α0-thalassemia/α+-thalassemia (-/-α or -/αTα). As a consequence of the relative excess of β-chains, individuals with HbH disease produce a variable amount of this abnormal hemoglobin, a tetramer of β-globin chains (β4). The HbH is unstable and precipitates inside the red cells and to some extent in erythroid precursors, causing membrane damage and premature erythrocyte destruction. As reported above, both hemolysis and ineffective erythropoiesis contribute to anemia in HbH disease, but the predominant mechanism is hemolysis. HbH has a much higher oxygen affinity than HbA and this may worsen the severity of anemia in patients with HbH disease. In the neonatal period, subjects with the HbH disease genotype have a consistently elevated Hb Bart (˜25%), which may still be detected in small amounts in some adults with HbH disease. The syndrome of HbH disease shows a considerable variability in clinical and hematologic severity. The majority of patients have minor disability, a few are severely affected requiring regular blood transfusions, and rare cases of HbH disease have been described with the hydrops fetalis clinical picture (see below).15, 65 The most relevant features are microcytic and hypochromic hemolytic anemia, hepatosplenomegaly, jaundice, and moderate thalassemia-like skeletal modifications.65, 237, 238, 239 The hemoglobin concentration is usually in the range of 7 to 10 g/dl and the MCV varies with age (being around 58 fl in childhood and around 64 fl in adulthood), while the MCH is around 18 pg irrespective of age. Reticulocytes range between 5% and 10%, and the α:β-globin chain synthesis ratio is markedly reduced, in the order of 0.20 to 0.60. Hemoglobin electrophoresis at alkaline pH shows a fast-moving band (HbH) in amounts ranging from 1% to 40%. Sometimes, because of the low quantity and the possible loss due to instability in the preparation of the hemolysate, HbH may escape detection. The most sensitive method to detect HbH consists of the incubation of peripheral blood cells for 1 to 2 hours at 37°C in the presence of supravital dyes (brilliant cresyl blue or methyl violet), which induce precipitation of the abnormal hemoglobin as inclusion bodies, easily recognizable at the microscope (Fig. 34.5). Determination of the α-globin genotype may be useful for prognosis of HbH disease, because the nondeletion forms are more severe than the deletion forms. Anemia is accentuated during pregnancy and may worsen quite dramatically with infections, fever, ingestion of oxidant drugs, aplastic anemia associated with Parvovirus B19, and hypersplenism.237 A variable spleen enlargement is almost always present, while liver enlargement is less common. A mild phenotype of HbH disease may result from the homozygous state for nondeletional α-thalassemia. Although the phenotype in some cases is closer to that of the homozygous state for α+-thalassemia, the degree of anemia and hypochromia may be more severe.250 In particular, homozygotes for the elongated α-chain variant Hb Constant Spring are asymptomatic, but show mild pallor and jaundice with liver and spleen enlargement in about 50% of the cases.15, 251 The hemoglobin level ranges from 9 to 11 g/dl and the MCV tends to be normal (88 ± 6 fl), while the MCH is slightly reduced (26 ± 3 pg). The peripheral blood contains HbA2, A, Hb Constant Spring, and traces of Hb Bart
rather than HbH.15 The severity of HbH disease shows a good correlation with the degree of α-chain deficiency. Thus, the more severe and variable phenotypes are associated with interactions involving nondeletion α-thalassemia defects that affect the dominant α2 gene, including (-/αConstant Springα), (-/αNcoIα) and (-/αHphIα).237, 238, 239 Patients with the nondeletional genotypes present earlier, with more severe hemolytic anemia, significant growth delay, dysmorphic facial features, and more marked hepatosplenomegaly; they require more transfusions. Few patients with HbH disease resulting from the interaction of α+-thalassemia (-α) with the deletion of the MCS regulatory region have been reported.66 A severe case of HbH disease due to deletions of variable extent of both upstream MCS-Rs while all four downstream α-globin genes are intact has been recently described.92
rather than HbH.15 The severity of HbH disease shows a good correlation with the degree of α-chain deficiency. Thus, the more severe and variable phenotypes are associated with interactions involving nondeletion α-thalassemia defects that affect the dominant α2 gene, including (-/αConstant Springα), (-/αNcoIα) and (-/αHphIα).237, 238, 239 Patients with the nondeletional genotypes present earlier, with more severe hemolytic anemia, significant growth delay, dysmorphic facial features, and more marked hepatosplenomegaly; they require more transfusions. Few patients with HbH disease resulting from the interaction of α+-thalassemia (-α) with the deletion of the MCS regulatory region have been reported.66 A severe case of HbH disease due to deletions of variable extent of both upstream MCS-Rs while all four downstream α-globin genes are intact has been recently described.92
 FIGURE 34.5. Hemoglobin H inclusion bodies. |
A very few cases of unusually severe HbH disease associated with hydrops fetalis due to coinheritance of α0– and α+-thalassemia have been described.65, 252, 253, 254 In four cases, the α+-thalassemia alleles were mutations of the α2 gene associated with hyperunstable α-globin variants. The interaction between two relatively common forms of α-thalassemia (-Med/αTSaudiα) can present with HbH hydrops fetalis.255 In these families, prenatal diagnosis can be indicated.
Patients with HbH disease may develop complications including hypersplenism, leg ulcers, gallstones, and abnormal left ventricular dysfunction. Hypersplenism has been reported in 10% of Thai patients with HbH disease, but seems to be rare elsewhere.256 Iron overload as assessed by serum ferritin is increased in a large proportion (50% to 75%) of patients and is significantly correlated with age.237, 239, 257 Some patients develop increased liver iron concentration.
In general, patients with HbH disease do not need any treatment. Some clinicians recommend folic acid supplementation as for other hemolytic anemias. Patients should be advised to avoid oxidant drugs because of the risk of hemolytic crisis. Occasional blood transfusions may be required when the hemoglobin level suddenly drops as a consequence of hemolytic or aplastic crisis. Pregnant women with HbH disease need careful monitoring of the hemoglobin levels. Splenectomy may be indicated in the presence of hypersplenism, but the potential complication of venous thrombosis, reported in some patients with HbH disease following splenectomy, should be considered.258, 259 Chelation therapy should be initiated in patients with elevated serum ferritin and/or liver iron concentration.
Hemoglobin Bart Hydrops Fetalis Syndrome
Hb Bart hydrops fetalis syndrome is the most severe α-thalassemia clinical condition, often associated with the absent function of all four α-globin genes (homozygous α0-thalassemia or -/-). A few cases of hydrops fetalis have been reported in infants with very low levels of α-chain synthesis, resulting from interaction of common α0-thalassemia determinants with uncharacterized nondeletion defects.253, 254, 256 Hb Bart hydrops fetalis syndrome is relatively common in Southeast Asia, while in Mediterranean populations it is relatively rare due to the low frequency of α0-thalassemia.15,260, 261 Because of the extreme rarity of the -/αα genotype, this disorder rarely, if ever, affects infants of African descent. A fetus homozygous for α0-thalassemia produces mainly Hb Bart (γ4), which is functionally useless for oxygen transport, and his or her survival to late pregnancy is due to the presence of small amounts of embryonic hemoglobins Portland 1 (ζ2γ2) and Portland 2 (ζ2β2). There is a marked variability in the intrauterine clinical course of fetuses with Hb Bart hydrops fetalis. Many pregnancies terminate unnoticed or early in gestation. In some cases pregnancy proceeds to term, but the fetus is stillborn or severely ill; in others the fetus does not become hydropic and is born normally.262, 263 The clinical features of this syndrome are those of a very severe anemia (Hb level range, 3 to 8 g/dl), with marked hepatosplenomegaly, generalized edema, signs of cardiac failure, and extensive extramedullary erythropoiesis in many organs.15, 264 Other congenital abnormalities, particularly of the skeletal, cardiovascular, and urogenital system, have been reported. Complications during pregnancy are common and include severe and mild pre-eclampsia (hypertension, fluid retention with or without proteinuria), polyhydramnios or oligohydramnios (increased or reduced accumulation of amniotic fluid, respectively), and antepartum hemorrhage. Postpartum complications include placenta retention, eclampsia (fits and coma), hemorrhage, anemia, and sepsis. At present there is no effective treatment for the Hb Bart hydrops fetalis syndrome. Early treatment with intrauterine transfusions after noninvasive monitoring by Doppler ultrasonography or in utero hematopoietic stem cell transplantation have been attempted, but may not be justified because of the unknown future risks for infants of severe developmental abnormalities.265, 266, 267, 268, 269 Given the severity of this syndrome and of the maternal obstetric complications, early termination of at-risk pregnancies is recommended and several regions have initiated universal prenatal screening programs to address homozygous α-thalassemia.270, 271
Unusual Forms of a-Thalassemia
There are two unusual forms of α-thalassemia: one is the acquired HbH disease associated with myelodysplasia, and other is the α-thalassemia associated with mental retardation syndrome.
α-Thalassemia/Myelodysplasia Syndrome (OMIM catalog #300448)
Patients with myelodysplasia may rarely develop an unusual form of HbH disease characterized by the presence of classic HbH inclusion bodies in red blood cells, often detectable levels of HbH (1% to 57%), and a severe microcytic and hypochromic anemia with anisopoikilocytosis.272 The α– to β-globin mRNA ratio, studied in a few patients, showed a marked reduction (0.06 to 0.50), and the α– to β-globin chain synthesis ratio was similarly reduced (α:β ratio = 0.28).273 Structural analysis of the α-globin genes and of their flanking regions has revealed no abnormalities in such patients.274 Recent studies have shown that some patients with α-thalassemia/myelodysplasia syndrome have point mutations and/or splicing abnormalities in the ATRX gene (see below).275 In one patient a large deletion of the telomeric region of the short arm of one allele of chromosome 16, including both α-globin genes, was reported.276
α-Thalassemia and Mental Retardation Syndromes
There are two different syndromes in which α-thalassemia is associated with mental retardation.235, 277 The first is characterized by a relatively mild mental retardation and a variety of facial and
skeletal abnormalities. These subjects have extended (1 to 2 megabases) deletions resulting from rearrangements of the short arm of chromosome 16. The deletions remove both α-globin genes and up to 52 other genes.278 Two common patterns of α-thalassemia have been described: One is characterized by parents with a normal α-globin genotype (αα/αα) whose affected offspring have the phenotype of severe α-thalassemia trait (genotype -/αα). In the other, one parent has the phenotype of the mild thalassemia trait and the child has HbH disease. This condition is called ATR-16 syndrome (OMIM catalog #141750).
skeletal abnormalities. These subjects have extended (1 to 2 megabases) deletions resulting from rearrangements of the short arm of chromosome 16. The deletions remove both α-globin genes and up to 52 other genes.278 Two common patterns of α-thalassemia have been described: One is characterized by parents with a normal α-globin genotype (αα/αα) whose affected offspring have the phenotype of severe α-thalassemia trait (genotype -/αα). In the other, one parent has the phenotype of the mild thalassemia trait and the child has HbH disease. This condition is called ATR-16 syndrome (OMIM catalog #141750).
The second group of patients has a complex phenotype characterized by severe mental retardation, quite uniform clinical features (hypertelorism, flat nasal bridge, triangular upturned nose, wide mouth, urogenital abnormalities), other developmental abnormalities, and defective α-globin synthesis, resulting in a relatively mild form of HbH disease. No structural changes of the α cluster or 16p chromosome have been found in these patients and the transmission is X-linked. Recently it has been shown that this syndrome is associated with mutations in an X-encoded gene, the ATRX gene, a member of the DNA helicase family.279, 280, 281 To date 128 acquired and/or inherited mutations predominantly lying in 2 highly conserved domains of the ATRX protein have been identified.282 ATRX, a large protein with 2492 residues, is a member of the snf2 family of ATP-dependent remodeling proteins and a key regulatory component of nucleosomal dynamics and higher order chromatin conformation. ATRX protein plays a prominent role in the control of gene transcription and in the maintenance of chromosome stability.283, 284 Mutations in this gene downregulate the expression of the α-globin genes and of other unidentified genes, producing the complex phenotype. This condition is referred to as Atrx syndrome (OMIM catalog #301040).
α-Thalassemia in Association with Structural Variants
A number of syndromes result from the interaction of α-thalassemia genes with those producing structurally abnormal hemoglobins. In some disorders, thalassemia genes that otherwise would have gone unnoticed are given clinical expression by the variant hemoglobin; in others, the relative amount of the variant hemoglobin is altered by the thalassemia gene. Features common to all these syndromes are red cell hypochromia and microcytosis, in addition to the presence of a hemoglobin variant. Some of the mutations causing α-chain structural variants appear to have occurred in chromosomes with only a single α-globin gene. Thus, Q/α0-thalassemia has a clinical phenotype similar to that of HbH disease.285 Affected subjects synthesize no HbA. This disorder has been described in individuals from Thailand, China, Iran, and India.286, 287 The mutation responsible for HbG-Philadelphia sometimes occurs on a chromosome with a single α-globin gene and other times on a chromosome containing both α genes. This variant is encountered primarily in black individuals.288 In persons with a normal α-globin gene on the same chromosome containing the HbG mutation, HbG-Philadelphia/α0-thalassemia (αGα/-) is characterized clinically by α-thalassemia minor; whereas in individuals with no normal α-gene cis to the αG-gene (-αG/-), the doubly heterozygous state resembles clinically HbH disease. The variant hemoglobin constitutes approximately 40% of the total concentration of hemoglobin in the former situation, and more than 90% in the latter.37 HbI/α-thalassemia has been reported in a black patient.289 That the gene for HbI is not linked in cis with an α-thalassemia gene is indicated by the presence of 30% HbA. The combination of α-thalassemia with β-chain variants is associated with a decrease in the relative amount of the variant hemoglobin and a clinical picture similar to that of the heterozygous state for the structural variant.15 The lower than usual percentage of the variant hemoglobin is attributed to the preferential binding of α-chains with βA-chains. The β-chain variants noted in association with α-thalassemia include HbS, HbC, HbE, and HbJ Bangkok.15 The interaction of α-thalassemia and the HbS trait produces a trimodal distribution in the relative amount of HbS. Individuals with a full complement of α-globin genes have more than 35% HbS, compared with 28% to 35% in those with the (-α/αα) genotype, 25% to 30% in those with the (-α/-α) genotype, and no more than 20% in those with the rare (-/-α) genotype.290, 291 Reductions in MCV and MCH are also observed. α-Thalassemia modifies some of the hematologic consequences of homozygous sickle cell anemia. Subjects with the (-α/-α) genotype have a higher hemoglobin concentration, lower red cell indices, fewer irreversibly sickled cells, a lower reticulocyte count, and lower serum bilirubin levels than subjects without concurrent α-thalassemia.292, 293, 294 The ameliorating effect of α-thalassemia is probably mediated by a decreased red cell concentration of HbS. α-Thalassemia fails, however, to temper significantly the clinical expression of sickle cell anemia. More information on the effects of α-thalassemia on sickle cell anemia can be found in Steinberg MH66 and in the chapter on sickle cell anemia in this book. For the interaction of α-thalassemia and HbE, see Hemoglobin E Syndromes in this chapter.
β-THALASSEMIA MAJOR
From a clinical point of view, the β-thalassemia syndromes represent the most relevant forms of thalassemia. The designations commonly used to describe the β-thalassemia syndromes are based on clinical severity. The most severe form is defined as β-thalassemia major and is characterized by transfusion-dependent anemia. Thalassemia intermedia is the term used to designate a form of anemia that, independently from the genotype, does not require transfusion, or only sporadic or intermittent transfusions. Thalassemia minor indicates the heterozygous state, which is usually completely asymptomatic. Thalassemia minima was used in the Italian literature to indicate a carrier in whom no hematologic or clinical symptoms were recognizable, but the term should probably be abandoned. Some authors use the term thalassemia minima to indicate the condition of silent carrier.
History
Originally, there was a disease called anemia splenica infantum that included several conditions, often not well distinguished from one another. Syphilis was considered a possible cause, as were tuberculosis and leishmaniosis. These children were usually born normally and grew normally until the second half of the first year, when they were noticed to become paler and paler, and to develop an enormous abdomen, containing a spleen that could extend from a few centimeters below the left costal margin to the iliac crest and below, sometimes visible from the outside. At this time in the disease course, the patients liked to be left alone and to lie down “in a monotonous morbid state.”28 Bone deformities, especially of the skull, soon appeared, giving the children a distinctive “Mongolian” appearance.295, 296 The disease was often present in more than one sibling; or, more frequently, other siblings had previously died of the same disease. The first systematic descriptions of what was going to be identified as thalassemia major came from Cooley and Lee295 from Michigan, who observed the disease in Italian and Greek children, and from Maccanti, a pediatrician from Ferrara, Italy, who also noted that the children were often coming from malarial areas near the Po river.28, 295 Anemia, leukocytosis, and normoblastemia were always present. Both groups tried unsuccessfully the entire armamentarium of therapies then available (arsenium, fresh veal bone marrow, sunshine, the
quartz lamp, cod liver oil, and, of course, iron) and even blood transfusions, which were helpful but short-lasting in one patient but caused increased hemolysis in another.28 This is not surprising, considering the very limited blood matching available at the time and the already enormous size of the spleen at presentation. Splenectomy and Roentgen irradiation of the spleen were also performed without benefit. All the children died shortly after presentation. Detailed autoptic data, showing peculiar abnormalities in the bones and spleen fibrosis, are available. Almost at the same time, Rietti, also from Ferrara, had reported three adult patients, two of whom were father and son, who presented with “primitive hemolytic jaundice” associated with decreased osmotic fragility.297 Anemia, microcytosis, anisocytosis, and basophilic stippling were noted. The syndrome was probably a form of thalassemia intermedia, and for a long time the eponymic title of Rietti-Greppi-Micheli was used in Italy, from the names of those who in those years described similar clinical pictures. In 1932, in consideration of the Mediterranean origin of the patients affected by Cooley anemia, Whipple and Bradford proposed the name of thalassemia, from the Greek word thalassa, meaning sea.298 Subsequently, the severe and the mild form of thalassemia were denominated thalassemia major and minor, respectively.299
quartz lamp, cod liver oil, and, of course, iron) and even blood transfusions, which were helpful but short-lasting in one patient but caused increased hemolysis in another.28 This is not surprising, considering the very limited blood matching available at the time and the already enormous size of the spleen at presentation. Splenectomy and Roentgen irradiation of the spleen were also performed without benefit. All the children died shortly after presentation. Detailed autoptic data, showing peculiar abnormalities in the bones and spleen fibrosis, are available. Almost at the same time, Rietti, also from Ferrara, had reported three adult patients, two of whom were father and son, who presented with “primitive hemolytic jaundice” associated with decreased osmotic fragility.297 Anemia, microcytosis, anisocytosis, and basophilic stippling were noted. The syndrome was probably a form of thalassemia intermedia, and for a long time the eponymic title of Rietti-Greppi-Micheli was used in Italy, from the names of those who in those years described similar clinical pictures. In 1932, in consideration of the Mediterranean origin of the patients affected by Cooley anemia, Whipple and Bradford proposed the name of thalassemia, from the Greek word thalassa, meaning sea.298 Subsequently, the severe and the mild form of thalassemia were denominated thalassemia major and minor, respectively.299
Unfortunately, the lack of communication between the two sides of the Atlantic made the research in this field, as in others, proceed slowly in parallel.300, 301 In 1940, Wintrobe reported the presence of a familial hemopoietic disorder in adolescents and adults of Italian origin, while in Italy, between 1943 and 1947, Silvestroni and Bianco defined the hematologic, clinical, and epidemiologic characteristics of thalassemia minor and its relationship with thalassemia major.302 A detailed report of that research can be found in a comprehensive book on thalassemia by Ida Bianco Silvestroni.303 The picture was further clarified by the identification of HbA2 and its increase in the parents of patients affected by thalassemia major.304, 305 The patients, on the other hand, were found to be completely devoid of HbA and to have, in addition to HbA2, only an alkali-resistant variant usually found in the newborn, HbF.306 The newly born erythrokinetic technique defined the enormous and ineffective erythropoiesis present in this disease.307 Between 1956 and 1961 the chemical structure of hemoglobin was described, and soon the complete sequences of globin chains were clarified.308
The idea of thalassemia resulting from a defect in the production of adult hemoglobin is due to the contribution of several authors. Globin chain synthesis was able to confirm this hypothesis.309 The developments in molecular biology are recent history and continue to clarify more aspects of the disease.
Clinical Features
The clinical picture of β-thalassemia major includes features that are due to the disease itself and others that represent the consequences of therapy and are, in a sense, iatrogenic.
Anemia
The early symptoms of the disease appear usually in the first year of life, at the time when the synthesis of γ-chains is not replaced by the synthesis of β-chains. In an ethnically composite population of transfusion-dependent children diagnosed in the United Kingdom, the mean age at presentation was reported to be 6 months, while in a study from Greece the age was 13.1 months, ranging from 2 to 36 months.310, 311 In a study from Sardinia the disease was recognized at around 8 months in patients with transfusion-dependent thalassemia but at age 2 years in non-transfusion-dependent children.312 The age at diagnosis is influenced by the molecular defect and by the degree of suspicion of the treating physician. Pallor is usually the first sign, accompanied by splenomegaly of various severity, fever, and failure to thrive.
 FIGURE 34.6. Radiograph of the skull. In the frontal area, the bone has a lamellated structure, parallel to the inner table of the diploe. In the parietal area, erythroid hyperplasia has perforated the outer table, producing a characteristic “hair on end” appearance. (Courtesy of Dr. C. Orzincolo.) |
Bone Deformities
Untransfused or poorly transfused patients with thalassemia develop typical bone abnormalities that were described even in the first reports of the disease and that are due to the extremely increased erythropoiesis, with consequent expansion of the bone marrow to 15 to 30 times normal. The skull is large and deformed by frontal and posterior bossing with the diploe increased in thickness to several times normal. The outer and inner tables are thin and the trabeculae are arranged in vertical striations, resulting in a “hair on end” appearance. A peculiar, stratified appearance of the skull has been reported (Fig. 34.6).313 The zygomatic bones are prominent, the base of the nose is depressed and pneumatization of the sinuses is delayed. Overgrowth of the maxilla produces severe malocclusion, with a rodent-like appearance. Metatarsal and metacarpal bones are the first to expand as a consequence of increased erythropoiesis (Fig. 34.7). The ribs are broad, often with a “rib-within-rib” appearance, and the vertebral bodies are square. The trabeculation of the medullary space gives the bones a mosaic pattern. Shortening of long bones is frequent, as a result of premature fusion of the humeral and femoral epiphyseal lines.314, 315 Extramedullary erythropoiesis gives rise to masses that protrude from bones where red marrow persists.316
Overgrowth from the vertebral bodies can cause cord compression and paraparesis.317 Ear impairment due to extramedullary marrow growing in the middle ear, and progressive visual loss caused by compressive optic neuropathy have been reported.318, 319 This kind of picture is more often present in patients with thalassemia intermedia, in whom transfusions are avoided at the price of intense autologous marrow hyperactivity. Improvement in the radiologic bone appearance in the cohorts of patients who have undergone regular transfusions from an early age has been striking. The lack of severe skull deformities is reflected in the mildness of thalassemic features that are now observed in most patients. However, bone lesions of a different nature are often observed as a consequence of excessive deferoxamine (DFO) therapy.
Osteoporosis
Reduced bone mineral density and consequent susceptibility to fractures have been observed in thalassemia patients and, in recent years, have been the subject of intense research. Mineral density is usually investigated with dual-energy x-ray absorptiometry (DEXA) at the spinal (L1 to L4) and femoral neck level. Osteoporosis is defined as a decrease in bone mineral density
≥2.5 SD below the young adult mean value, while a decrease between -1 and -2.5 SD is defined as osteopenia.320 Osteoporosis in thalassemia has been found to affect 48% of the patients, with an additional 44% affected by osteopenia.321 Although more frequent and severe in males than in females, this complication represents an important cause of morbidity in adult patients of both sexes.321 The pathogenesis of osteoporosis in thalassemia major is multifactorial and results from a variety of genetic and acquired factors. The polymorphism at the Sp1 site of the collagen type I gene (COLIA 1) has been associated with severe osteoporosis and pathologic fractures of the spine and the hip.321, 322, 323 Moreover, the vitamin D receptor (VDR) Bsm1 and Fok1 polymorphisms were found to be risk factors for bone mineral damage, low bone mineral density, and short stature in prepubertal and pubertal patients.324 However, different studies of genetic polymorphisms have given contradictory results.322, 325 Acquired factors include the primary disease itself, causing ineffective hematopoiesis with progressive bone marrow expansion; and several secondary factors such as endocrine dysfunction, iron overload and chelation therapy, vitamin deficiencies, and decreased physical activity.326, 327 In particular, vitamin D deficiency is frequent among adolescents.328 Male sex, lack of spontaneous puberty, and diabetes represent significant risk factors for osteoporosis, while transfusional history, chelation, and erythropoietic activity do not.329
≥2.5 SD below the young adult mean value, while a decrease between -1 and -2.5 SD is defined as osteopenia.320 Osteoporosis in thalassemia has been found to affect 48% of the patients, with an additional 44% affected by osteopenia.321 Although more frequent and severe in males than in females, this complication represents an important cause of morbidity in adult patients of both sexes.321 The pathogenesis of osteoporosis in thalassemia major is multifactorial and results from a variety of genetic and acquired factors. The polymorphism at the Sp1 site of the collagen type I gene (COLIA 1) has been associated with severe osteoporosis and pathologic fractures of the spine and the hip.321, 322, 323 Moreover, the vitamin D receptor (VDR) Bsm1 and Fok1 polymorphisms were found to be risk factors for bone mineral damage, low bone mineral density, and short stature in prepubertal and pubertal patients.324 However, different studies of genetic polymorphisms have given contradictory results.322, 325 Acquired factors include the primary disease itself, causing ineffective hematopoiesis with progressive bone marrow expansion; and several secondary factors such as endocrine dysfunction, iron overload and chelation therapy, vitamin deficiencies, and decreased physical activity.326, 327 In particular, vitamin D deficiency is frequent among adolescents.328 Male sex, lack of spontaneous puberty, and diabetes represent significant risk factors for osteoporosis, while transfusional history, chelation, and erythropoietic activity do not.329
 FIGURE 34.7. Mosaic pattern produced by trabeculation in the bones of the hand of a patient with thalassemia major. Note the rectangular contour of the metacarpals. |
Defective osteoblastic activity is thought to be the major pathogenetic mechanism for osteoporosis. There are data demonstrating in thalassemia patients increased serum levels of Dickkopf-1, a soluble inhibitor of osteoblast differentiation that correlates with reduced bone mineral density. Also sclerostin, an inhibitor of osteoblast function, is increased and correlates with bone mineral density of the spine, radius, and femoral neck.330 In addition, there is evidence of increased osteoclast activation. Elevated markers of bone resorption, such as urinary N-terminal peptides of collagen type I and serum tartrate-resistant acid phosphatase isoform 5b, have been demonstrated.331, 332 The increased osteoclast activity seems to be due to an overproduction of cytokines that are involved in osteoclast differentiation and function.333 There is evidence that the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) pathway mediates osteoclast proliferation in thalassemia and contributes to the pathogenesis of osteoporosis.334 The hypothesis that the RANKL/OPG system is involved in mediating the action of sex steroids on bone334 has not been confirmed.335 Fractures, often secondary to mild or moderate trauma, are more frequent in thalassemia patients than in the general population. In a retrospective study, 12% of patients with thalassemia major were found to have suffered from fractures, with an equal distribution between males and females. Prevalence increased with age. The presence of other endocrinopathies, anthropometric parameters, heart disease, or hepatitis C were not significant independent predictors of fractures.336, 337 Bone pain of varying severity is a common complaint among adult patients and has been attributed to expanded bone marrow with consequent pressure on the cortical bone.338 Magnetic resonance imaging (MRI) in these cases may show the reappearance of hypercellular areas in bones previously replaced by fatty marrow. Reduced and irregular mineralization of the bone has been found using microradiography and X-rays in thalassemic patients with and without clinically evident bone abnormalities.339 Back pain is sometimes associated with compression fractures and intervertebral disc degeneration.327, 340
In a recent study, pain was found to be associated with low vitamin D, lower bone density, and bisphosphonate use.341 Osteoporosis is a progressive disease, thus early detection, prevention, and treatment are essential for effective control of this potentially debilitating condition.326 Annual follow-up should be started during adolescence.342 Therapy includes sex hormone replacement therapy, regular exercise, and a diet rich in calcium and vitamin D. In consideration of the pathogenetic data suggesting that in thalassemia patients the reduced osteoblastic activity is accompanied by a comparable or even greater increase in bone resorption, antiresorptive drugs such as bisphosphonates are being increasingly used. To date, alendronate, pamidronate, and zoledronate have been reported to be effective in increasing bone mineral density and normalizing bone turnover.343, 344, 345 Neridronate has improved bone mineral density and reduced back pain in a cohort of thalassemia patients with osteoporosis.346 One study evaluating the effect of calcitonin on bone mass showed that it prevented bone pain, improved radiologic findings, and decreased the number of fractures.347 Other agents, like teriparatide and strontium ranelate, are being studied, but their effects remain to be proven.342
Cholelithiasis
Gallstones have been reported in patients with thalassemia348, 349 (Fig. 34.8). The percentages found were variable, depending on the transfusion regimens and consequent residual inefficient erythropoiesis and hemolysis, on the time of splenectomy, and, more importantly, on the associated presence of the (TA)7 promoter mutation of the gene of uridindiphosphoglucoronyl-transferase.350 A recent cooperative study including 858 patients with transfusion-dependent thalassemia found a prevalence of cholelithiasis of 30%. The Gilbert genotype [homozygosity for (TA)7 motif] influenced both the prevalence of cholelithiasis and the age at which it developed.351 Ultrasonography of the gallbladder should be checked regularly. If gallstones are present at the time of splenectomy, cholecystectomy should be performed at the same time.
Thromboembolic Complications
Numerous reports of thromboembolic complications in thalassemia have been published. In a multicenter study the
frequency of thromboembolic events was found to be 4% in patients with thalassemia major and 10% in patients with thalassemia intermedia.352 Other groups have reported similar prevalences.353 In a large study of 8860 patients from different countries, female sex, previous splenectomy, and profound anemia were found to represent risk factors.354 A chronic hypercoagulable state has been observed even in childhood.205 The mechanisms underlying hypercoagulability in thalassemia are still unclear. Several authors197, 200 have suggested that the presence of a chronic hypercoagulable state could be due to the procoagulant effect of the anionic phospholipids exposed on the surface of the damaged circulating red blood cells and to endothelial derangement occurring as a consequence of an inflammatory state associated with the disease. Also, blood cells and platelets have an important role in increasing the thrombotic risk in thalassemia.355
frequency of thromboembolic events was found to be 4% in patients with thalassemia major and 10% in patients with thalassemia intermedia.352 Other groups have reported similar prevalences.353 In a large study of 8860 patients from different countries, female sex, previous splenectomy, and profound anemia were found to represent risk factors.354 A chronic hypercoagulable state has been observed even in childhood.205 The mechanisms underlying hypercoagulability in thalassemia are still unclear. Several authors197, 200 have suggested that the presence of a chronic hypercoagulable state could be due to the procoagulant effect of the anionic phospholipids exposed on the surface of the damaged circulating red blood cells and to endothelial derangement occurring as a consequence of an inflammatory state associated with the disease. Also, blood cells and platelets have an important role in increasing the thrombotic risk in thalassemia.355
 FIGURE 34.8. Spine of a 35-year-old patient with thalassemia major. Reduced mineral bone density is evident, in addition to a gallstone-filled gallbladder. |
In addition, vascular endothelial cell injury and the peroxidative status due to iron overload are possible pathogenetic mechanisms.198, 206, 356
Concomitant prothrombotic conditions are frequently present in thalassemia patients after the first decade of life: insulindependent diabetes, estrogen therapy, atrial fibrillation, and postsplenectomy thrombocytosis, among others. Prophylactic measures are necessary when additional risk factors (e.g., surgery, parturition, immobilization) are present.354 (See also thalassemia intermedia.)
Pseudoxanthoma Elasticum
Pseudoxanthoma Elasticum (PXE) is an autosomal recessive multisystem disorder affecting elastic tissues. In the majority of families affected by PXE the gene carrying mutations is the ABCC6 gene, encoding a transmembrane transporter protein probably involved in calcium and phosphate homeostasis and primarily expressed in the liver, the kidneys, and the intestine. More than 300 mutations have been identified and lead to the absence of a functional ABCC6 protein. This absence results in a deficiency of circulating factors which should prevent aberrant mineralization, and induces the accumulation of calcium phosphate in skin, eye, and vascular lesions.357, 358 Clinically it is characterized by typical lesions of the skin (small yellowish papules or larger coalescent plaques), eyes (breaks of the elastic lamina of Bruch’s membrane called angioid streaks), and arteries (degeneration of the elastic lamina of the arterial wall often accompanied by arterial calcification).359, 360 An acquired PXE-like syndrome has been described in several hemolytic disorders.361, 362 The first reports of typical skin lesions, angioid streaks in the retina, calcified arterial walls, and aortic valve disease in two patients affected by thalassemia was published by Aessopos et al. in 1989.363 Several subsequent reports have confirmed the existence of a clinical syndrome resembling PXE in thalassemia patients.364, 365, 366, 367 It appears to be age-dependent and it is more common in thalassemia intermedia than in thalassemia major. In a study published in 1998 and including patients affected by thalassemia intermedia older than 30 years, arterial calcifications were found in 55%, skin lesions in 20%, and ocular alterations in 52%. Eighty-five percent had at least one of the three typical lesions.368
In PXE-like syndromes, lesions are structurally indistinguishable from those of the inherited form. The typical histopathologic features are the abnormal, mineralized, and fragmented elastic fibers (elastorrhexia) in skin, eyes, and arterial blood vessels. Nevertheless, β-thalassemia patients do not harbour mutations in the ABCC6 gene,366, 369 and the pathophysiology remains unclear. At first, it was suggested that, like so many other complications of the disease, this syndrome could be the result of an iron-induced oxidative tissue damage, caused by hemolysis and iron overload.368, 370 Recently, it has been demonstrated that a significant, progressive, liver-specific downregulation of Abcc6 expression can be found in the mouse model of β-thalassemia. Despite the fact that these mice do not develop connective mineralization, they could represent a model for clarifying the increased susceptibility to dystrophic mineralization in dermal, ocular, and vascular tissue of β-thalassemia patients.371 The abnormalities in elastic fibers of arterial walls may lead to several serious and life-threatening vascular complications in both the inherited and acquired forms. In the thalassemic population several cases of cardiac involvement have been reported, including rupture of chordae tendineae and aneurysmatic dilatation of the ascending aorta.368, 372, 373, 374, 375 In an Italian study, five out of 14 thalassemia patients with a PXE-like syndrome died because of cardiovascular complications. Thrombotic events and gastrointestinal and intracranial bleeding, which preclude the use of platelet antiaggregants, can also complicate the clinical course.376 Close surveillance of these patients is therefore mandatory.
No effective therapy is available for PXE and PXE-like syndromes. Several promising avenues for the treatment are currently being explored. The phosphate binders, a group of drugs given in the attempt to limit the intestinal absorption of phosphate, and consequently normalizing the serum calcium phosphate product, could offer a means of reducing the calcium/phosphate load in patients with PXE. Histopathologic regression of skin calcifications was demonstrated in 3 out of six patients treated with aluminum hydroxide for 1 year, and in all six no progression of the ocular angioid streaks was observed.377 In a knocked-out ABCC6 gene (Abcc6 -/-) mouse model of PXE, a magnesium carbonate-enriched diet completely prevented mineralization of the vibrissae,
an early biomarker of the mineralization process.378 The mechanisms by which magnesium may prevent calcium phosphate deposition in tissues are not clear, but a marked increase in the urinary output of calcium, with concomitant reduction in phosphate, has been obtained in these mice.378, 379
an early biomarker of the mineralization process.378 The mechanisms by which magnesium may prevent calcium phosphate deposition in tissues are not clear, but a marked increase in the urinary output of calcium, with concomitant reduction in phosphate, has been obtained in these mice.378, 379
These results suggest that changes in diet, and specifically changes in dietary magnesium and phosphate binders, may offer a potential treatment modality for this so far intractable disease.380 Recent reports on the use of intraocular injection of antiangiogenic agents targeting vascular endothelial growth factor in age-associated macular degeneration have suggested that significant improvements in visual acuity could also be achieved in patients with PXE.381, 382
Molecular strategies based on specific mutations identified in the ABCC6 gene, gene therapy approaches targeting a transgene to restore its function in the liver, and anti-mineralization factors with a potential role as modifiers (fetuin-A, for example) are still being tested.
Secondary Gout
Hyperuricemia is not unusual in thalassemia patients, but gouty arthritis has rarely been reported.383
Laboratory Findings and Diagnosis
Laboratory data at presentation are characterized by HbF levels ranging from 10% to 100%; HbA2 may be normal or increased up to 5% to 7%; and the remaining percentage is constituted by HbA. HbF is heterogeneously distributed among red cells. The reticulocyte count is low, usually below 1%. MCV is typically 60 to 70 fl and MCH is 12 to 18 pg/cell. In the peripheral blood smear a great variation in size and shape of the erythrocytes is always evident. Microcytes, tear drop cells, and nucleated red cells are present, together with large and pale target cells (Fig. 34.9). The Hb composition varies mainly accordingly to the patient’s genotype. Homozygotes for β0-thalassemia mutations have only HbF (93% to 97%) and HbA2 (3% to 7%) and no HbA, while homozygotes for β+-thalassemia mutations and β0/β+ genetic compounds have HbA, HbA2, and a variable, but always significantly elevated, amount of HbF (10% to 90%). HbA2 in homozygous β-thalassemia may be normal or increased and so of no diagnostic value. Hb separation to determine the hemoglobin pattern can be performed with different methods (electrophoresis at alkaline or acidic pH, isoelectric focusing, high performance liquid chromatography [for details, see (384, 385)]). In the case of transfused patients, diagnosis can be made by globin chain synthesis analysis from peripheral blood reticulocytes, which shows a severe α/non-α imbalance (usually higher than 2), or by β-globin gene analysis to identify the various DNA mutations (details on DNA methods can be found in specific surveys and laboratory manuals).386
 FIGURE 34.9. Peripheral blood smears in β-thalassemia major (A) and intermedia (B) and in heterozygous β-thalassemia (C). |
Therapy
Blood Transfusion
The decision on whether and when to start transfusion therapy in a child with thalassemia is not an easy one and should be based on a precise molecular diagnosis indicating the severity of the thalassemic defect, on the level of hemoglobin, and on general conditions and satisfactory growth. Evidence of increasing splenomegaly, of bony expansion, and of consequent modification of facial features should also be considered. The age at which starting transfusion becomes necessary in thalassemia varies according to the prevalent genotypes in a particular ethnicity. Delaying the age of transfusion increases the risk of developing allo- and autoantibodies.223
For many years after the description of thalassemia major as a clinical entity, therapy was limited to blood transfusion when symptoms of anemia were so severe as to incapacitate the patient. The corresponding levels of hemoglobin (Hb) were different in different patients, but varied between 6 and 7 g/dl. As a consequence of continuous anemia, erythropoiesis, although inefficient, was intense, the bone marrow underwent an enormous expansion, and the plasma volume increased greatly. In addition, the liver and spleen increased in size as a consequence
of both extramedullary erythropoiesis and hemolytic activity in the reticuloendothelial tissue. The bone deformities caused by the expanded marrow are typical of thalassemia and gave to all the poorly transfused patients similar features (see above). In the 1960s, however, the superiority of regular and methodically repeated transfusions was recognized, first by Orsini in France, and later by Wolman in Philadelphia and Piomelli in New York, who started a program of chronic transfusion directed at maintaining a baseline Hb level adequate to eliminate hypoxia and thus suppress its consequences.387, 388, 389 It was calculated then that the amount of iron administered to maintain a minimum Hb of 9.5 g/dl was only 50% greater than that resulting from a baseline Hb of 6 g/dl, and that the additional iron intake could be counterbalanced in part by the reduction of intestinal iron absorption. This kind of regimen, that never allowed Hb level to fall below 9.5 to 10 g/dl, was termed “hypertransfusion.” Complete bone marrow suppression, however, is seldom obtained at these Hb levels, and therefore some bone remodeling and expansion of the blood volume persist. To completely correct the effects of anemia, Propper et al. in 1980 launched what was called a “supertransfusion” regimen, where the pretransfusion hematocrit was kept at ≥ 35%.390 The hypothesis was that, as a consequence of the reduction of the blood volume, the amount of blood needed to maintain a higher baseline would not have been greater than the blood volume used for the lower baseline. A few papers from Europe confirmed the data; but, since the blood that is destroyed between transfusions and that needs replacing is a percentage of the patient’s red cell mass, patients kept at a higher baseline Hb level require a larger amount of blood and therefore accumulate more iron.391, 392, 393 In a study of patients kept at a pretransfusion hemoglobin level between 9 and 10 g/dl, the erythroid marrow activity, evaluated through the measurement of serum transferrin receptor, did not exceed two to three times normal levels. On the basis of these studies, the majority of centers choose to transfuse at a Hb level of 9 to 10.5 g/dl.394 The recommended post transfusion Hb is 14 to 15 g/dl. Leuko-reduced packed red cells are recommended for eliminating the adverse reactions attributed to contaminating white cells and for preventing platelet alloimmunization. The number of residual leukocytes should not be higher than 1 × 106. At present the preferred method for leukoreduction is prestorage filtration of whole blood with an inline filter within 8 hours after blood collection. Alternatively, laboratory filtration can be used pretransfusion. With this method, packed red blood cells are prepared from donor whole blood, then filtered prior to release from the blood bank. Finally, the packed red cell unit can be filtered at the bedside.
of both extramedullary erythropoiesis and hemolytic activity in the reticuloendothelial tissue. The bone deformities caused by the expanded marrow are typical of thalassemia and gave to all the poorly transfused patients similar features (see above). In the 1960s, however, the superiority of regular and methodically repeated transfusions was recognized, first by Orsini in France, and later by Wolman in Philadelphia and Piomelli in New York, who started a program of chronic transfusion directed at maintaining a baseline Hb level adequate to eliminate hypoxia and thus suppress its consequences.387, 388, 389 It was calculated then that the amount of iron administered to maintain a minimum Hb of 9.5 g/dl was only 50% greater than that resulting from a baseline Hb of 6 g/dl, and that the additional iron intake could be counterbalanced in part by the reduction of intestinal iron absorption. This kind of regimen, that never allowed Hb level to fall below 9.5 to 10 g/dl, was termed “hypertransfusion.” Complete bone marrow suppression, however, is seldom obtained at these Hb levels, and therefore some bone remodeling and expansion of the blood volume persist. To completely correct the effects of anemia, Propper et al. in 1980 launched what was called a “supertransfusion” regimen, where the pretransfusion hematocrit was kept at ≥ 35%.390 The hypothesis was that, as a consequence of the reduction of the blood volume, the amount of blood needed to maintain a higher baseline would not have been greater than the blood volume used for the lower baseline. A few papers from Europe confirmed the data; but, since the blood that is destroyed between transfusions and that needs replacing is a percentage of the patient’s red cell mass, patients kept at a higher baseline Hb level require a larger amount of blood and therefore accumulate more iron.391, 392, 393 In a study of patients kept at a pretransfusion hemoglobin level between 9 and 10 g/dl, the erythroid marrow activity, evaluated through the measurement of serum transferrin receptor, did not exceed two to three times normal levels. On the basis of these studies, the majority of centers choose to transfuse at a Hb level of 9 to 10.5 g/dl.394 The recommended post transfusion Hb is 14 to 15 g/dl. Leuko-reduced packed red cells are recommended for eliminating the adverse reactions attributed to contaminating white cells and for preventing platelet alloimmunization. The number of residual leukocytes should not be higher than 1 × 106. At present the preferred method for leukoreduction is prestorage filtration of whole blood with an inline filter within 8 hours after blood collection. Alternatively, laboratory filtration can be used pretransfusion. With this method, packed red blood cells are prepared from donor whole blood, then filtered prior to release from the blood bank. Finally, the packed red cell unit can be filtered at the bedside.
The current recommended practice is to use red cell units that have not been stored more than 2 weeks. Extended red cell antigen typing, including at least the Rh antigens, Duffy, Kidd, and Kell, is recommended before the patient is started on a transfusion regimen. Transfusion of young red cells (neocytes) obtained by centrifugation has been proposed in the attempt to reduce the total blood requirement, but the results obtained were not sufficient to justify the increased cost and the exposure to a larger number of donors.395, 396
In general, the transfusion rate is 5 to 6/ml/kg/hour. In the case of patients with cardiac failure, blood should be infused at a slower rate (no more than 3 to 4 ml/Kg/h), and the administration of diuretics before transfusion is advised. The recommended interval between transfusions should take into account the patient’s practical needs, as long as a pretransfusional Hb ranging between 9 and 10.5 g/dl is maintained. It is important to keep an accurate record of the amount transfused, in order to calculate the iron intake of the patient. The annual intake is expressed in ml/kg/yr of pure red cells, assuming that 1 ml of pure red cells contains 1.08 mg of iron.397 It has been observed that the level of Hb maintained during the warm months is lower than that during the cold months. Possible mechanisms include expansion of plasma volume with resultant hemodilution in the patient, and lower hemoglobin content in donor blood.398
For the comfort and safety of the patient, there should be a designated area at the hospital where transfusions will be administered and supervised by regular staff, well known to the patients and their families. It is often necessary for the center to be able to provide after hours transfusions, especially for children going to school and for working patients.
Complications of Transfusions
Although blood transfusions are life-saving for thalassemia patients, who no longer die of anemia, they can be complicated by transfusion reactions, alloimmunization, infections, and hemosiderosis.
Febrile Nonhemolytic Transfusion Reactions. A cooperative effort conducted 20 years ago, the Cooleycare initiative, reviewing more than one hundred thousand red cell transfusions in Italy and Greece, found that transfusion reactions complicated 1% of all transfusional events in 16% of the patients.399 About 90% of the red cell units infused were leukocyte-poor. Chills, fever, urticaria, headache, and chest pain accounted for more than 80% of symptoms reported, and in two-thirds of cases, reactions were reported during transfusion.400 Alloimmunization to HLA-antigens on leukocytes is the most common cause of febrile reaction in multiply transfused thalassemic patients. If blood is not filtered prestorage, cytokines may develop during storage and be responsible for the reaction. Treatment includes acetaminophen or hydrocortisone. Allergic reactions, due to plasma proteins and manifesting as hives, pruritus, and more rarely edema, are best treated with an antihistaminic drug.
Alloimmunization. Alloimmunization and autoimmunization can complicate transfusion therapy. The frequency of alloimmunization against red cells is variable, the lower percentages being found in patients who received blood matched for the AB0, Rhesus, and Kell systems from their first transfusion. In a multicenter study, allo- and autoantibodies were reported in 16.5% and 4.9% of patients, respectively. Splenectomized patients were 2.5 times more likely to have developed alloantibodies.401
Another study from a single large center found that 19.5% of the thalassemia patients developed alloantibodies, 94% of them being against the Rhesus or Kell antigens. Older age, higher transfusion frequency, and splenectomy were risk factors for alloimmunization.402
The risk of developing alloantibodies is not uniform and is probably genetically determined. Transfusion in infancy seems to induce immune tolerance.223 Asians appear to be at a higher risk of developing allo- and autoantibodies.403 In addition, the risk of alloimmunization is higher in individuals from ethnic minorities that, in general, donate less. In the US, the donation rates of African-American are 25% to 50% of that of white individuals.404
This phenomenon is present also in Europe, where the immigrant population, potentially at risk for hemoglobinopathies, is increasing in recent years. Donation by minorities should be encouraged to prevent the formation of red blood cell alloantibodies, which can result in hemolytic transfusion reactions and difficulty in finding appropriate red blood cells for future transfusions.
Infections. The risk of transfusion-transmitted viral infection is well known. Among the most frequent and clinically relevant are the widely diffused hepatotropic viruses hepatitis B and C (HBV, HCV ). The prevalence of infection of these viruses in multitransfused patients is different in different parts of the world and is directly related to the frequency in that population. Worldwide, from 0.3% to 5.7% of thalassemia patients are hepatitis B surface antigen (HBsAg)-positive405, 406, 407 and from 4.4% to 85% are positive for anti-hepatitis C antibodies.408, 409 The prevalence of HBV chronic infection is higher in countries in Asia and Southeast
Asia, whereas HCV chronic infection is widespread throughout the world. The DNA-recombinant vaccine against hepatitis B virus, safe and effective, is available and should be administered to all patients who have not yet been infected. Hepatitis G virus and GB virus C (GBV-C) are RNA viruses that were independently identified in 1995, and were subsequently found to be two isolates of the same virus. Together with transfusion-transmitted (TT) virus, they are common among thalassemia patients but have not been found to contribute to chronic hepatocellular damage.410 West Nile Virus infection has become of concern in recent years. Epidemics have been reported, and the virus can be transmitted through blood transfusion.411, 412 In the United States, testing for West Nile virus antibodies has been implemented in 2003,413 and nucleic acid testing is widely used in Europe in endemic areas.
Asia, whereas HCV chronic infection is widespread throughout the world. The DNA-recombinant vaccine against hepatitis B virus, safe and effective, is available and should be administered to all patients who have not yet been infected. Hepatitis G virus and GB virus C (GBV-C) are RNA viruses that were independently identified in 1995, and were subsequently found to be two isolates of the same virus. Together with transfusion-transmitted (TT) virus, they are common among thalassemia patients but have not been found to contribute to chronic hepatocellular damage.410 West Nile Virus infection has become of concern in recent years. Epidemics have been reported, and the virus can be transmitted through blood transfusion.411, 412 In the United States, testing for West Nile virus antibodies has been implemented in 2003,413 and nucleic acid testing is widely used in Europe in endemic areas.
CMV is widespread in most populations. A European collaborative study revealed a positive CMV IgG test in two-thirds of the thalassemia patients examined.414
HIV infection was acquired almost exclusively before the systematic screening of blood donation. In 1987 the prevalence of HIV in thalassemia patients from 13 European or Mediterranean countries was found to be 1.56%. Two years later no HIV seroconversion was observed in the same areas when a total of 2,972 patients affected by thalassemia who had received 96,518 blood units were examined.415 Since 2004, several cases of transfusion-associated variant Creutzfeld-Jacobs Disease (vCJD) have been reported and linked to blood collected from preclinically affected donors. Animal data suggest that all blood components are vectors for prion disease transmission.416 Malaria can be transmitted by transfusion in endemic areas.417 A screening program for Trypanosoma cruzi, a parasitic infection endemic in Central and South America, which is spreading into nonendemic countries with the migration of infected individuals, was developed in 1998 in the United Kingdom418 and in 2007 in the United States.413 Although significant improvements have been made to further decrease the incidence of transfusion-transmitted infections, risks remain for infectious disease agents specific to red blood cell concentrates including emerging viruses, bacteria, protozoa, and residual contaminating leukocytes.419, 420, 421 Pathogen and leukocyte inactivation of red blood cells is therefore of potential relevance in order to transfuse patients with completely pathogen-inactivated blood units. A pathogen inactivation system for red blood cells, based on a nucleic acid (DNA and RNA) targeting and crosslinking compound, is being actively developed and is reaching clinical application.422
In a recent review, the major results from more than 150 articles published by the Retrovirus Epidemiology Donor Study (REDS), conducted from 1989 to 2001, and the REDS-II, conducted from 2004 to 2012, were reported. These were National Heart, Lung, and Blood Institute-funded, multicenter programs focused on improving blood safety and availability in the United States. REDS-II also included Brazil and China, while the just launched REDS-III will also include South Africa. The three major research domains of REDS/REDS-II have been evaluation of infectious risk, blood donation availability, and blood donor characterization. Blood safety studies have included protocols evaluating epidemiologic and/or laboratory aspects of human immunodeficiency virus, human T-lymphotropic virus 1/2, hepatitis C virus, hepatitis B virus, West Nile virus, cytomegalovirus, human herpesvirus 8, parvovirus B19, malaria, Creutzfeldt-Jakob disease, influenza, and Trypanosoma cruzi infections.423
Predisposing factors for bacterial infections include prior splenectomy, iron overload, and use of the iron chelator deferoxamine. Despite the availability of prophylactic measures against encapsulated organisms, the risk of severe, sometimes fatal, infections is still high in splenectomized patients.424 Iron overload and deferoxamine favor the growth of organisms such as Yersinia enterocolitica and Klebsiella pneumoniae, Yersinia being more prevalent in temperate regions and Klebsiella in tropical and subtropical areas. Yersinia infection should be suspected in the presence of fever, diarrhea, right-lower-quadrant abdominal pain, and a palpable abdominal mass. Abdominal suppurative complications have been reported.425 Deferoxamine, but not deferiprone or deferasirox, increases growth of Yersinia both in vivo and in vitro. In fact, being a siderophore, deferoxamine can be used by this organism as a source of iron. Growth of Klebsiella, on the contrary is only moderately enhanced by DFO in vitro and not at all by deferiprone or deferasirox. Several reports have described infection with Aeromonas hydrophila in Asia. The growth of Aeromonas hydrophila, however, does not seem to be affected by any of the three chelators.426
Hemosiderosis
In patients who undergo transfusional therapy for several years, the accumulation of iron, if untreated, causes considerable morbidity and ultimately leads to death. Since each unit of blood contains approximately 200 milligrams of iron, a patient who receives 25 to 30 units of blood a year, by the third decade of life, in the absence of chelation, will accumulate over 70 g of iron.427 In addition to the iron administered through blood transfusions, a hyperactive bone marrow will favor increased intestinal iron absorption that will contribute, although marginally, to the total body load. This mechanism has recently been clarified. Duodenal iron absorption is regulated by the hepcidin-ferroportin axis, and hepcidin, in turn, is regulated by plasma iron concentration and iron stores. Hepcidin is a 25-amino acid peptide, synthesized in the hepatocytes, that controls the concentration of ferroportin on the intestinal epithelium. Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates ferroportin activity by inducing its internalization and degradation. Low levels of hepcidin correlate with higher levels of ferroportin, resulting in increased intestinal iron absorption.
In addition, hepcidin is homeostatically regulated by the iron requirements of erythroid precursors for hemoglobin synthesis. In iron-loading anemias associated with ineffective erythropoiesis, hepcidin production is suppressed by a signal mediated by growth differentiation factor 15 (GDF15), which is expressed at high levels in conditions of intense erythropoietic drive.430 As a consequence, in β-thalassemia, whenever the bone marrow is not completely suppressed by blood transfusions, iron absorption is increased, even in the presence of iron overload.195, 428, 429, 430, 431
Excessive iron may damage the cell by several mechanisms. In patients who have fully saturated transferrin, a significant fraction of the total iron in plasma circulates in the form of low molecular weight complexes not bound to transferrin.432 Although the exact mechanism of tissue damage remains unclear, the most important pathogenetic factor appears to be iron-induced peroxidative injury to the phospholipids of lysosomes and mitochondria. The redox active component of non-transferrin-bound iron is referred to as the labile plasma iron and it can be identified with oxidantsensitive fluorescent methods.433, 434, 435
Control of circulating labile plasma iron is crucial to prevent oxidative damage and to decrease the risk of organ dysfunction. At present, an easy method for serial labile plasma iron measurements is not available.436, 437
Excessive iron stores lead to depletion of substances that defend against free radical attack, e.g., among others, ascorbic acid, which is oxidized to oxalate, and vitamin E.438 This in turn causes sequestration of the iron in the reticuloendothelial system, somehow protecting tissues from siderosis.439 At suboptimal concentration, ascorbic acid is a pro-oxidant and enhances the catalytic effect of iron in free radical formation. The presence of the genetic hemochromatosis mutations does not seem to influence the degree of iron overload and its consequences in regularly transfused and chelated patients with thalassemia major.232
Assessment of Iron Stores. The iron status of multitransfused patients can be assessed by several methods. The use of two or more parameters will usually provide a good estimate of the total amount of iron accumulated. Serum iron is always elevated. Transferrin saturation correlates reasonably well with serum ferritin.440, 441 After only a few years of transfusion, however, transferrin is completely saturated in the majority of patients. In thalassemia major, serum ferritin has, in general, been found to correlate well with iron stores, as measured by phlebotomy, and with liver iron, either measured directly by liver biopsy or by MRI.195, 429 (Fig. 34.10) Significantly higher ferritin levels are present in patients with endocrinopathies,442, 443 cardiac failure, and arrhythmias than in patients without such complications. Levels above 2,500 ng/dl have been reported to be associated with a four-fold higher risk of death.444
Several variables can interfere with the reliability of ferritin as a marker of iron overload. Ferritin, being an acute phase reactant, is increased in chronic disease, malignancy, or inflammatory disorders. A ferritin concentration of 4,000 mcg/L is considered the maximum level of physiologic synthesis, while higher values would represent the release of intracellular ferritin from damaged cells.445 Ascorbic acid deficiency can lead to decreased synthesis and release of ferritin. This can therefore lead to ferritin levels that are only mildly elevated, even in the presence of massive iron stores.446 A low level of hepcidin also results in iron depletion of macrophages, decreasing their secretion of ferritin and, therefore, serum ferritin levels. This phenomenon is particularly evident in thalassemia intermedia.195 Conversely, patients with active liver disease may have high levels of serum ferritin that do not mirror the body iron load.447. Despite all this, serial measurements of serum ferritin remain a reliable means, and the easiest one, to evaluate iron overload and efficacy of chelation therapy. The measurement of iron excretion over 24 hours, after an intramuscular injection of 500 mg of desferrioxamine was, in the past, used largely to establish the time to start chelation therapy or to evaluate the iron burden. Unfortunately, the correlation between urinary excretion and body iron is not very good, as many factors, including dose of chelator administered per unit of weight of the patient and vitamin C status, contribute to influence iron excretion.
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