++
Once cloning and sequencing of globin genes from patients with many different forms of thalassemia were possible, the wide spectrum of mutations underlying these conditions became clear. A picture of remarkable heterogeneity has emerged. For more extensive coverage of this topic, the reader is referred to several monographs and reviews.7,9,10,44,45,46
++
β-thalassemia is extremely heterogeneous at the molecular level.7 More than 200 different mutations have been found in association with the β-thalassemia phenotype.7 Broadly, they fall into deletions of the β-globin gene and nondeletional mutations that may affect the transcription, processing, or translation of β-globin messenger (Table 48–2 and Fig. 48–5). Each major population group has a different set of β-thalassemia mutations, usually consisting of two or three mutations forming the bulk and large numbers of rare mutations. Because of this distribution pattern, only approximately 20 alleles account for the majority of all β-thalassemia determinants (see Fig. 48–1).
++
++
++
At least 17 different deletions affecting only the β genes have been described. With one exception, the deletions are rare and appear to be isolated, single events. The 619-bp deletion at the 3′ end of the β gene is more common,48 but even that is restricted to the Sind and Gujarati populations of Pakistan and India, where it accounts for approximately 50 percent of β-thalassemia alleles.48 The Indian 619-bp deletion removes the 3′ end of the β gene but leaves the 5′ end intact. Many of the other deletions remove the 5′ end of the gene and leave the δ gene intact.49,50,51,52,53 Homozygotes for these deletions have β0-thalassemia. Heterozygotes for the Indian deletion have increased hemoglobin A2 and F levels identical to those seen in heterozygotes for the other common forms of β-thalassemia. Heterozygotes for the other deletions all have unusually high hemoglobin A2 levels.7 Increased δ-chain production results from increased δ-gene transcription in cis to the deletion, possibly as a result of reduced competition from the deleted 5′ β gene for transcription factors.
+++
Other Transcriptional Mutations
++
Several different base substitutions involve the conserved sequences upstream from the β-globin gene.7 In every case, the phenotype is β+-thalassemia, although considerable variability exists in the clinical severity associated with different mutations of this type. Several mutations, at positions –88 and –87 relative to the mRNA CAP site, for example,54,55 are close to the CCAAT box, whereas others lie within the TATA box homology.56,57,58,59
++
Some mutations upstream from the β-globin gene are associated with even more subtle alterations in phenotype. For example, a C→T substitution at position –101, which involves one of the upstream promoter elements, is associated with “silent” β-thalassemia, that is, a completely normal (“silent”) phenotype that can be identified only by its interaction with more severe forms of β-thalassemia in compound heterozygotes.60 A single example of an A→C substitution at the CAP site (+1) was described in an Asian Indian who, despite being homozygous for the mutation, appeared to have the phenotype of the β-thalassemia trait.61
++
Upstream regulatory mutations confirm the importance of the role of conserved sequences in this region as regulators of the transcription of the β-globin genes and provide the basis for some of the mildest forms of β-thalassemia, particularly those in African populations, and for some varieties of “silent” β-thalassemia.
+++
RNA-Processing Mutations
++
One surprise about β-thalassemia has been the remarkable diversity of the single-base mutations that can interfere with the intranuclear processing of mRNA.
++
The boundaries of exons and introns are marked by invariant dinucleotides, GT at the 5′ (donor) and AG at the 3′ (receptor) sites. Single-base changes that involve either of these splice junctions totally abolish normal RNA splicing and result in the β0-thalassemia phenotype.7,62,63,64,65,66
++
Highly conserved sequences involved in mRNA processing surround the invariant dinucleotides at the splice junctions. Different varieties of β-thalassemia involve single-base substitutions within the consensus sequence of the IVS-1 donor site.55,58,63,64,65,66,67,68,69 These mutations are particularly interesting because of the remarkable variability in their associated phenotypes. For example, substitution of the G in position 5 of IVS-1 by C or T results in severe β+-thalassemia.55 On the other hand, a T→C change at position 6, found commonly in the Mediterranean region,70 results in a very mild form of β+-thalassemia. The G→C change at position 5 has also been found in Melanesia and appears to be the most common cause of β-thalassemia in Papua New Guinea.71
++
RNA processing is affected by mutations that create new splice sites within either introns or exons. Again, these lesions are remarkably variable in their phenotypic effect, depending on the degree to which the new site is utilized compared with the normal splice site. For example, the G→A substitution at position 110 of IVS-1, which is one of the most common forms of β-thalassemia in the Mediterranean region, leads to only approximately 10 percent splicing at the normal site and hence results in a severe β+-thalassemia phenotype.72,73 Similarly, a mutation that produces a new acceptor site at position 116 in IVS-1 results in little or no β-globin mRNA production and the β0-thalassemia phenotype.74 Several mutations that generate new donor sites within IVS-2 of the β-globin gene have been described.55,68
++
Another mechanism for abnormal splicing is activation of donor sites within exons (Fig. 48–6). For example, within exon 1 is a cryptic donor site in the region of codons 24 through 27. This site contains a GT dinucleotide. An adjacent substitution that alters the site so that it more closely resembles the consensus donor splice site results in its activation, even though the normal site is active. Several mutations in this region can activate this site so that it is utilized during RNA processing, with the production of abnormal mRNAs.75,76,77,78 Three of the substitutions—A→G in codon 19, G→A in codon 26, and G→T in codon 27—result in reduced production of β-globin mRNA and an amino acid substitution so that the mRNA that is spliced normally is translated into protein. The abnormal hemoglobins produced are hemoglobins Malay, E, and Knossos, respectively, all of which are associated with a β-thalassemia phenotype, presumably as a result of reduced overall output of normal mRNA (Fig. 48–6). A variety of other cryptic splice mutations within introns and exons have been described.44
++
++
Another class of processing mutations involves the polyadenylation signal site AAUAAA in the 3′ untranslated region of β-globin mRNA.79,80,81 For example, a T→C substitution in this sequence leads to only one-tenth the normal amount of β-globin mRNA and hence the severe β+-thalassemia phenotype.79
+++
Mutations Causing Abnormal Translation of Messenger RNA
++
Base substitutions that change an amino acid codon into a chain termination codon, that is, nonsense mutations, prevent translation of the mRNA and result in β0-thalassemia. Many substitutions of this type have been described.7,44 For example, a codon 17 mutation is common in Southeast Asia,82,83 and a codon 39 mutation occurs at a high frequency in the Mediterranean region.84,85
++
The insertion or deletion of one, two, or four nucleotides in the coding region of the β-globin gene disrupts the normal reading frame and results, upon translation of the mRNA, in the addition of anomalous amino acids until a termination codon is reached in the new reading frame. Several frameshift mutations of this type have been described.7,44 Two mutations—the insertion of one nucleotide between codons 8 and 9 and a deletion of four nucleotides in codons 41 and 42—are common in Asian Indians.63 The latter deletions are found frequently in different populations in Southeast Asia.83
++
An unusual β+-thalassemia was described in a patient from the Czech Republic in whom a full-length L1 transposon was inserted into the second intron of β-globin, creating a β+-thalassemia phenotype by an undefined molecular mechanism.86
+++
Dominantly Inherited β-Thalassemia
++
Families in which a picture indistinguishable from moderately severe β-thalassemia has segregated in mendelian dominant fashion have been reported sporadically.87,88 Because this condition often is characterized by the presence of inclusion bodies in the red cell precursors, it has been called inclusion body β-thalassemia. However, because all severe forms of β-thalassemia have inclusions in the red cell precursors, the term dominantly inherited β-thalassemia is preferred.7,89 Sequence analysis has shown that these conditions are heterogeneous at the molecular level, but that many involve mutations of exon 3 of the β-globin gene. The mutations include frameshifts, premature chain termination mutations, and complex rearrangements that lead to synthesis of truncated or elongated and highly unstable β-globin gene products.7,89,90,91,92,93 The most common mutation of this type is a GAA→TAA change at codon 121 that leads to synthesis of a truncated β-globin chain.94 Although an abnormal β-chain product from loci affected by mutations of this type is unusual, many of these conditions are designated as hemoglobin variants.
++
The reason why mutations occurring in exons 1 and 2 produce the classic form of recessive β-thalassemia whereas the bulk of the dominant thalassemias result from mutations in exon 3 has become clearer. In the former case, very little abnormal β-globin mRNA is found in the cytoplasm of the red cell precursors, whereas exon 3 mutations are associated with full-length but abnormal mRNA accumulation. The different phenotypes of these premature termination codons have been suggested to reflect a phenomenon called nonsense-mediated RNA decay, a surveillance system to prevent transport of mRNA coding for truncated peptides. Presumably this process is active in the case of exon 1 or 2 mutations, in which affected mRNAs are degraded, but is not active in the case of exon 3 mutations.95,96,97 A complete list of the mutations that underlie the dominant β-thalassemias is given in reference 44.
+++
Unstable β-Globin Variants
++
Some β-globin chain variants are highly unstable but are capable of forming a viable tetramer. The resulting unstable hemoglobins may precipitate in the red cell precursors or in the blood, giving rise to a spectrum of conditions ranging from dominantly inherited β-thalassemia to a hemolytic anemia similar to the anemia associated with other unstable hemoglobins. The first unstable hemoglobin to be described was hemoglobin Indianapolis.98 Its structure was characterized by DNA analysis performed on stored autopsy material; however, the original description proved to be incorrect.99
++
A number of extremely mild β-thalassemia alleles are either silent or almost unidentifiable in heterozygotes (see Table 48–2). Some alleles are in the region of the promoter boxes of the β-globin gene, but others involve the CAP sites or the 5′ or 3′ untranslated regions.7,44 These alleles usually are identified by finding a form of β-thalassemia intermedia in which one parent has a typical thalassemia trait and the other parent appears to be normal but, in fact, is a carrier of one of the mild β-thalassemia alleles.
+++
β-Thalassemia Mutations Unlinked to the B-Globin Gene Cluster
++
Several family studies suggest the existence of mutations that result in the β-thalassemia phenotype but do not segregate with the β-globin genes100; however, their molecular basis has not been determined. Further evidence for the existence of novel mutations of this type can be found in reference 7.
+++
Variant Forms of β-Thalassemia
++
In several forms of β-thalassemia, the hemoglobin A2 level is normal in heterozygotes. Some cases result from “silent” β-thalassemia alleles, whereas others reflect the coinheritance of β- and δ-thalassemia.7
++
The δβ-thalassemias are classified into the (δβ)+- and (δβ)0-thalassemias (Table 48–3). The (δβ)0-thalassemias are further divided into (δβ)0-thalassemia, in which both the δ- and β-globin genes are deleted, and (Aγδβ)0-thalassemia, in which the Gγ, δ, and β genes are deleted. Because many different deletion forms of δβ-thalassemia have been described, they are further classified according to the country in which they were first identified (Table 48–3).
++
+++
(δβ)0- and (Aγδβ)0-Thalassemia
++
Nearly all these conditions result from deletions involving varying lengths of the β-globin gene cluster. Many different varieties have been described in different populations (see Table 48–3), although their heterozygous and homozygous phenotypes are very similar.7 Rare forms of these conditions result from more complex gene rearrangements. For example, one form of (Aγδβ)0-thalassemia, found in Indian populations, does not result from a simple linear deletion but rather from a complex rearrangement with two deletions, one affecting the Aγ gene and the other the δ and β genes. The intervening region is intact but inverted.101 Figure 48–7 illustrates some of these conditions.
++
++
The (δβ)+-thalassemias usually are associated with the production of structural hemoglobin variants called Lepore.102 Hemoglobin Lepore contains normal α chains and non-α chains that consist of the first 50 to 80 amino acid residues of the δ chains and the last 60 to 90 residues of the normal C-terminal amino acid sequence of the β chains. Thus, the Lepore non-α chain is a β-fusion chain. Several different varieties of hemoglobin Lepore have been described—Washington-Boston, Baltimore, and Hollandia—in which the transition from δ to β sequences occurs at different points.7 The fusion chains probably arose by nonhomologous crossing over between part of the δ locus on one chromosome and part of the β locus on the complementary chromosome (Fig. 48–8). This event results from misalignment of chromosome pairing during meiosis so that a δ-chain gene pairs with a β-chain gene instead of with its homologous partner.103 Figure 48–8 shows such a mechanism should give rise to two abnormal chromosomes: the first, the Lepore chromosome, will have no normal δ or β loci but simply a δβ fusion gene. Opposite the homologous pairs of chromosomes should be an anti-Lepore (βδ) fusion gene and normal δ and β loci. A variety of anti–Lepore-like hemoglobins have been discovered, including hemoglobins Miyada, P-Congo, Lincoln Park, and P-Nilotic.7 All the hemoglobin Lepore disorders are characterized by a severe form of δβ-thalassemia. The output of the γ-globin genes on the chromosome with the δβ fusion gene is not increased sufficiently to compensate for the low output of the δβ fusion product. The reduced rate of production of the δβ fusion chains of hemoglobin Lepore presumably reflects the fact that its genetic determinant has the δ gene promoter region, which is structurally different from the β-globin gene promoter and is associated with a reduced rate of transcription of its gene product.
++
+++
δβ-Thalassemia-Like Disorders Resulting from Two Mutations in the β-Globin Gene Cluster
++
A heterogeneous group of nondeletion δβ-thalassemias has been described, most resulting from two mutations in the εγδβ-globin gene cluster (see Table 48–3). Strictly speaking, they are not all δβ-thalassemias, but they often appear in the literature under this title because their phenotypes resemble the deletion forms of (δβ)0-thalassemia. In the Sardinian form of δβ-thalassemia, the β-globin gene has the common Mediterranean codon 39 nonsense mutation that leads to an absence of β-globin synthesis. The relatively high expression of the Aγ gene in cis gives this condition the δβ-thalassemia phenotype because of a point mutation at position –196 upstream from the Aγ gene (see “Hereditary Persistence of Fetal Hemoglobin” below). The phenotypic picture, in which heterozygotes have 15 to 20 percent hemoglobin F and normal hemoglobin A2 levels, is identical to that of δβ-thalassemia.103 Another condition having the β-thalassemia phenotype, with greater than 20 percent hemoglobin F in heterozygotes, has been described in a Chinese patient in whom defective β-globin chain synthesis appears to result from an A→G change in the ATA sequence in the promoter region of the β-globin gene.104 The increased γ-chain synthesis, which appears to involve both Gγ and Aγ cis to this mutation, remains unexplained. A disorder originally called δβ-thalassemia has been described in the Corfu population.105,106 The condition results from two mutations in the β-globin gene cluster: first, a 7201-bp deletion that starts in the δ-globin gene, IVS-2, position 818 to 822, and extends upstream to a 5′ breakpoint located 1719 to 1722 bp 3′ to the ψβ-gene termination codon; and second, a G→A mutation at position 5 in the donor site consensus region of IVS-1 of the β-globin gene. The output from this chromosome consists of relatively high levels of γ chains with very low levels of β chains. The condition resembles δβ-thalassemia in the homozygous state, with almost 100 percent hemoglobin F, traces of hemoglobin A, but no hemoglobin A2. Heterozygotes have only slightly elevated hemoglobin F levels, with a phenotype similar to “normal A2β-thalassemia.”
++
These rare conditions107,108,109,110,111,112,113 result from long deletions that begin upstream from the β-gene complex 55 kb or more 5′ to the ε gene and terminate within the cluster (see Fig. 48–7). In two cases, designated Dutch110,111 and English,112 the deletions leave the β-globin gene intact, but no β-chain production occurs even though the gene is expressed in heterologous systems.
++
The molecular basis for inactivation of the β-globin gene cis to these deletions was clarified by the discovery of the LCR approximately 50 kb upstream from the εγδβ-globin gene cluster (see “Genetic Control and Synthesis of Hemoglobin” above). Removal of this critical regulatory region seems to completely inactivate the downstream globin gene complex. The Hispanic form of εγδβ-thalassemia113 results from a deletion that includes most of the LCR, including four of the five DNase-1-hypersensitive sites. These lesions appear to close down the chromatin domain that usually is open in erythroid tissues and delay replication of the β-globin genes in the cell cycle. Thus, although they are rare, the lesions have been of considerable importance because analysis of the Dutch deletion first pointed to the possibility of a major control region upstream from the β-like-globin gene cluster and ultimately led to the discovery of the β-globin LCR.
+++
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
++
This heterogeneous group of conditions produces phenotypes very similar to those of the δβ-thalassemias, except that defective β-chain production appears to be almost, but in some forms not completely, compensated by persistent γ-chain production. These conditions are best classified into deletion and nondeletion forms (Table 48–4). In the past, the conditions were classified into pancellular and heterocellular varieties, depending on the intercellular distribution of fetal hemoglobin. However, this subdivision now appears to bear little relevance to their molecular basis and probably relates more to the particular level of fetal hemoglobin and how its cellular distribution is determined.7
++
++
The deletion forms of HPFH are heterogeneous (see Fig. 48–7). The two African varieties result from extensive deletions of similar length (<70 kb) but with staggered ends, differing phenotypically only in the proportions of Gγ and Aγ chains produced.114 Another type of HPFH results from misalignment during crossing over between the Aγ- and β-globin genes, resulting in production of Aγβ fusion genes (see Fig. 48–8) that combine with α chains to form the hemoglobin variant called hemoglobin Kenya.115,116 Hemoglobin Kenya is associated with an increased output of hemoglobin F, although at a lower level than in the deletion forms of HPFH. A theory that adequately explains the phenotypic differences between δβ-thalassemia and the deletion forms of HPFH has not been developed.7
++
The nondeletion determinants of HPFH can be classified into those that map within the β-globin gene cluster and those that segregate independently. The former are subdivided into Gγβ+ and Aγβ+ varieties, indicating persistent Gγ- or Aγ-chain synthesis in association with β-globin production directed by the β gene cis (on the same chromosome) to the HPFH determinant. Analysis of the overexpressed γ genes revealed in each case a single-base substitution in the region immediately upstream from the transcription start site.7,117,118,119,120 Clustering of these substitutions and lack of similar changes in normal γ genes suggest they are responsible for persistent hemoglobin F production (Fig. 48–9). This region of DNA likely is involved in binding of trans-acting proteins involved in the normal developmental repression of γ-gene expression, either by decreasing the affinity for an inhibitory factor normally present in adult life or by increasing the affinity for a factor promoting gene expression. The most common of these conditions are Greek Aγβ+ HPFH and a form of Gγβ+ HPFH, which has been found in several different African populations. If the upstream point mutations associated with persistent γ-chain production occur on the same chromosome as β-globin genes that carry β0-thalassemia mutations, the clinical phenotype is converted from HPFH to δβ-thalassemia, albeit with different hemoglobin A2 levels.
++
++
In some cases, other nondeletional forms of HPFH have been related to small structural changes in the β-globin gene cluster (see Table 48–4). Although strictly speaking not a true form of HPFH, because even in homozygotes it may not be associated with increased hemoglobin F levels, the T→C polymorphism at position –158 to the Gγ-globin gene121 might be associated with an increased output of hemoglobin F under conditions of erythropoietic stress.
++
Other forms of HPFH are characterized by the persistence of low levels of fetal hemoglobin production distributed in a heterocellular manner. In all populations studied, a small proportion of individuals have an increased amount of hemoglobin F and F cells, that is, red cells that can be detected when blood films are treated with antibodies against hemoglobin F. Although this condition originally was called the Swiss form of HPFH because it was first recognized in Swiss army recruits,122 it is observed in every racial group. Using a variety of genetic approaches, it has become clear that a number of genes may be involved in the generation of heterocellular HPFH, including loci at Xp22.2-p22.3,6q23,8q, and 2p15123,124,125,126,127,128; the latter linkage has been identified as the oncogene BCL11A. The mechanism whereby these different loci affect the level of F cells in normal individuals and increase their levels in conditions like thalassemia and sickle cell anemia remain to be determined, but their coinheritance with these conditions may have an extremely beneficial effect of their associated phenotypes.129
++
Several point mutations and deletions that reduce δ-globin synthesis have been described. They are summarized in reference 7.
++
Table 48–5 summarizes the different classes of α-thalassemia mutations. The α-globin gene haplotype can be written αα, indicating the α1 and α2 genes, respectively. A normal individual has the genotype αα/αα. A deletion involving one (–α) or both (– –) α genes can be further classified based on its size, written as a superscript; thus, –α3.7 indicates a deletion of 3.7 kb including one α gene. When the sizes of the deletions are not established, a superscript describing their geographic or family origin is useful; thus, – – MED describes a deletion of both α genes first identified in individuals of Mediterranean origin. In thalassemia haplotypes in which both genes are intact, that is, nondeletion lesions, the nomenclature αNDα is given, with the superscript ND indicating the gene is thalassemic. However, when the precise molecular defect is known, as in hemoglobin Constant Spring, for example, αNDα can be replaced by the more informative αCSα. The molecular pathology and population genetics of the α-thalassemias have been the subject of several extensive reviews.7,41,45,130,131
++
++
Many deletions that involve both α genes, and therefore abolish α-chain production from the affected chromosome, have been described (Fig. 48–10).7 Several of the 3′ breakpoints fall within a 6- to 8-kb region at the 3′ end of the α-globin complex, suggesting this represents a breakpoint cluster region with a high level of recombination.132 In at least five of the deletions, the 5′ breakpoints also appear to cluster. This gives rise to a situation in which the 5′ breakpoints are located approximately the same distance apart and in the same order along a chromosome as their respective 3′ breakpoints. It is possible that such staggered deletions arise from illegitimate recombination events that delete an integral number of chromatin loops as they pass through their nuclear attachment points during replication. This mechanism has also been suggested to underlie some of the deletion forms of HPFH. One of these deletions (– –MED) involves a more complex rearrangement that introduces a new piece of DNA bridging the two breakpoints in the α-gene cluster. This new sequence originates upstream from the α cluster and appears to have been replicated into the junction in a manner suggesting that the upstream segment of DNA also lies at the base of a replication loop. At least some of these deletions seem to have arisen by recombination events between Alu repeat sequences.
++
++
Several other mechanisms for the generation of α0-thalassemia have been identified. In one case of unusual genetic interest, a long (>18 kb) deletion that removes the α1 gene and the region downstream was identified in which the α2 gene remains intact but is completely inactivated, giving the α0-thalassemia phenotype. Although the inactive α2 gene retains all its local and remote cis-regulatory elements, its expression is completely silenced and its CpG island is completely methylated as a result of transcription of antisense RNA expressed from a locus that had been juxtaposed to the α2 gene because of the large deletion.133,134 In some cases, this condition results from a terminal truncation of the short arm of chromosome 16 to a site 50 kb distal to the α-globin genes.135 It is interesting that the telomeric consensus sequence (TTAGGGG)n has been added directly to the site of the break. Because this mutation is stably inherited, telomeric DNA alone appears sufficient to stabilize the broken chromosome end. This observation raises the possibility that other genetic diseases result from chromosomal truncations.
++
Several deletions have been identified that appear to downregulate α-globin genes by removing the α-globin LCR (HS40).7,136,137 In each case, the α-globin genes are left intact, although in one the 3′ breakpoint is found between the ξ and ψξ genes, thus removing the ξ gene. These deletions appear to completely inactivate the α-globin gene complex, just as deletions of the β-globin LCR inactivate the entire β-gene complex. Such deletions have not been observed in the homozygous state, presumably because they would be lethal.
+++
α+-Thalassemia Gene Deletions
++
The most common forms of α+-thalassemia (–α3.7 and –α4.2) involve deletion of one or the other of the duplicated α-globin genes (see Figs. 48–10 and 48–11).
++
++
Each α gene is located within a region of homology approximately 4 kb long, interrupted by two nonhomologous regions. The homologous regions are believed to have resulted from an ancient duplication event and to have subsequently subdivided, presumably by insertions and deletions, to give three homologous subsegments referred to as X, Y, and Z (see Fig. 48–11). The duplicated Z boxes are 3.7 kb apart, and the X boxes are 4.2 kb apart. Misalignment and reciprocal crossover between these segments at meiosis can give rise to chromosomes with either single (–α) or triplicated (ααα) α-globin genes. Such an occurrence between homologous Z boxes deletes 3.7 kb of DNA (rightward deletion). A similar crossover between the two X blocks deletes 4.2 kb of DNA (leftward deletion –α4.2).138 The corresponding triplicated α-gene arrangements are referred to as αααanti–3.7 and αanti–4.2.139–141 More detailed analysis of these crossover events indicates they occur more commonly in the Z box. At least three different –α3.7 deletions have been found, depending on exactly where the crossover occurred.142 These deletions are designated –α3.7I, –α3.7II, and –α3.7III, respectively. Other, rarer deletions of a single α gene have been observed.7
+++
Nondeletion α-Thalassemia
++
Because expression of the α2 gene is two to three times greater than expression of the α1 gene, the finding that most of the nondeletion mutants discovered to date affect predominantly α2 gene expression is not surprising. Presumably this is ascertainment bias because of the greater phenotypic effect of these lesions. It also is possible that defective expression of the α2 gene has come under greater selective pressure.
++
Like the β-thalassemia mutations, α-thalassemia mutations7 can be classified according to the level of gene expression they affect (see Table 48–5). Several processing mutations have been identified. For example, a pentanucleotide deletion includes the 5′ splice site of IVS-1 of the α2-globin gene. This mutation involves the invariant GT donor splicing sequence and thus completely inactivates the α2 gene.143 A second mutant of this type, found commonly in the Middle East, involves the poly-A addition signal site (AATAAA→AATAAG) and downregulates the α2 gene by interfering with 3′ end processing.144,145
++
A second group of nondeletion α-thalassemias results from mutations that interfere with translation of mRNA.7 Several mutations involve the initiation codon.146,147,148,149 In one case, for example, the initiation codon is inactivated by a T→C transition.146 In another case, efficiency of initiation is reduced by a dinucleotide deletion in the consensus sequence around the start signal.149 Five mutations that affect termination of translation and give rise to elongated α chains have been identified: hemoglobins Constant Spring, Icaria, Koya Dora, Seal Rock, and Pakse.7 Each mutation specifically changes the termination codon TAA so that an amino acid is inserted instead of the chain terminating (Fig. 48–12). This process is followed by read-through of mRNA that is not normally translated until another “in-phase” stop codon is reached. Thus, each of these variants has an elongated α chain. The “read-through” of α-globin mRNA that usually is not utilized likely reduces its stability.150 Several nonsense mutations occur, for example, one in exon 3 of the α2-globin gene.151 Finally, several mutations occur that cause α-thalassemia by producing highly unstable α-globin chains, including hemoglobins Quong Sze,152 Suan Doc,153 Petah Tikvah,154 and Evanston.155 A complete list of nondeletion α-thalassemia alleles is given in reference 45.
++
+++
Interactions of α-Thalassemia Haplotypes
++
Many α-thalassemia haplotypes have been described, and potentially more than 500 interactions are possible!7 These phenotypes result in four broad categories: (1) normal, (2) conditions characterized by mild hematologic changes but no clinical abnormality, (3) hemoglobin H disease, and (4) hemoglobin Bart’s hydrops fetalis syndrome. The heterozygous states for deletion or nondeletion forms of α+-thalassemia either cause extremely mild hematologic abnormalities or are completely silent. In populations where α-thalassemia is common, the homozygous state for α+-thalassemia (–α/–α) can produce a hematologic phenotype identical to that of the heterozygous state for α0-thalassemia (– –/αα), that is, mild anemia with reduced mean cell hemoglobin and mean cell volume values.
++
Hemoglobin H disease usually results from the compound heterozygous state for α0-thalassemia and either deletion or non-deletion α+-thalassemia. It occurs most frequently in Southeast Asia (– –SEA/–α3.7) and the Mediterranean region (usually – –MED/–α3.7).
++
The hemoglobin Bart’s hydrops fetalis syndrome usually results from the homozygous state for α0-thalassemia, most commonly – –SEA/– –SEA or – –MED/– –MED. A few infants with this syndrome who synthesized very low levels of α chains at birth have been reported. Gene-mapping studies suggest these cases result from interaction of α0-thalassemia with nondeletion mutations (ααND).
+++
Unusual Forms of α-Thalassemia
++
Some unusual forms of α-thalassemia are completely unrelated to the common forms of the disease that occur in tropical populations. These conditions, which can occur in any racial groups, include α-thalassemia associated with mental retardation or leukemia. Their importance lies with the diagnostic problems they may present and, more importantly, the light that elucidation of the α-thalassemia pathology may shed on broader disease mechanisms.
+++
Molecular Pathology of the α-Thalassemia Mental Retardation Syndrome
++
The first descriptions of noninherited forms of α-thalassemia associated with mental retardation suggested the lesions involving the α-globin gene locus were acquired in the paternal germ cells and that their molecular pathology might help elucidate the associated developmental changes.156 Two separate syndromes of this type now are evident. In one group of patients, long deletions involve the α-globin gene cluster and remove at least 1 Mb.157 This condition can arise in several ways, including unbalanced translocation involving chromosome 16, truncation of the tip of chromosome 16, and loss of the α-globin gene cluster and parts of its flanking regions by other mechanisms. These findings localize a region of approximately 1.7 Mb in band 16p13.3 proximal to the α-globin genes as being causative of mental handicap.41
++
The second group is characterized by defective α-globin synthesis associated with severe mental retardation and a relatively homogeneous pattern of dysmorphology.158 Extensive structural studies have shown no abnormalities of the α-globin genes. These chromosomes direct the synthesis of normal amounts of α-globin in mouse erythroleukemia cells, suggesting that α-thalassemia results from deficiency of a trans-activating factor involved in regulation of the α-globin genes. This condition is encoded by a locus on the short arm of the X chromosome.159 ATR-X, the gene involved, is a DNA helicase with many features of a DNA-binding protein. Many different mutations of this gene have been identified in different families with the ATR-X (α-thalassemia X-linked mental retardation) syndrome.131,160 Studies have identified a plant homeodomain (PHD) region and an adenosine triphosphatase (ATPase)/helicase domain.161 Because patients with ATR-X syndrome show defective methylation of recombinant DNA arrays and related defects, this condition likely is one of a growing list of disorders that result from disordered chromatin remodeling.162,163
+++
α-Thalassemia and Myelodysplasia
++
The hematologic findings of hemoglobin H disease or mild α-thalassemia occasionally are observed in elderly patients with myeloid leukemia or the myelodysplastic syndrome. Earlier studies suggested this finding resulted from an acquired defect of α-globin synthesis in which the α-globin genes were completely inactivated in the neoplastic hemopoietic cell line.164 The molecular basis for this observation now is known to reside in a variety of different mutations involving ATR-X.41,165 The relationship of these somatic mutations of ATR-X to the neoplastic transformation remains to be determined. The molecular defect of other cases of acquired α-thalassemia, such as that seen in variable combined immunodeficiency,166 also remains to be defined.