Intervening Sequences or Introns

A remarkable finding was made on initial study of the globin genes: the coding region, rather than being organized in a single continuous unit, is interrupted by noncoding DNA known as intervening sequences (IVSs), or introns. Most eukaryotic genes contain one or more introns. As indicated in Figure 21-4 , globin genes are interrupted at two positions. The discontinuous nature of the coding region of globin genes poses a formidable problem for the formation of mRNA that must be translated into globin polypeptides on cytoplasmic ribosomes. Transcription of a globin gene generates a precursor mRNA containing introns. Formation of mature mRNA is accomplished by posttranscriptional processing, termed RNA splicing . The pathway of RNA processing is depicted in Figure 21-5 .

Structure of the human α- and β-globin genes. Untranslated (UT) regions, exon, and intervening sequences (IVS, introns) are depicted by green, salmon, and blue boxes , respectively.

Expression of the β-globin gene. Transcription of the gene generates the precursor ribonucleic acid (RNA), which is processed by RNA splicing to form messenger RNA.

RNA splicing must be executed with exquisite precision for functional mRNA to be generated. Because translation of mRNA proceeds by the reading of triplets (codons), excision of introns needs to be accurate to the nucleotide; otherwise, shifts in the reading frame of the translated polypeptide will result. RNA processing is guided by specific sequences, known as splice site consensus sequences, located at the 5′ and 3′ boundaries of the introns. The donor site, which marks the 5′ end of the intron, generally conforms to the sequence 5′ (C/A)AG′ GT (A/G)AGT, where the prime sign indicates the position of splicing and GT is an essentially invariant dinucleotide at the 5′ end of the intron. The acceptor site, which defines the 3′ end of the intron, usually fits the consensus 5′ (T/C) _n N(C/T) AG ′G, where n is 11 or greater, N is any nucleotide, the prime sign indicates the site of splicing, and AG is an essentially invariant dinucleotide. Excision of introns generally occurs between the dinucleotides GT and AG, known as the GT-AG rule.

Conserved Features of Mature Globin mRNA

Sequences of human globin genes represented in processed globin mRNA include additional segments located before (5′) and after (3′) the coding region. These untranslated regions are depicted in Figure 21-5 . In addition, the mature mRNA is modified at both termini. At the 5′ end, a methylated guanylic acid (m ⁷ G) cap structure is present. A variable number of adenylic acid residues are added at the 3′ end and constitute a poly(A) tail. The 5′ cap structure appears to be important for efficient initiation of mRNA translation, whereas the poly(A) tail contributes to mRNA stability. Overlapping the beginning of the mRNA sequence in genomic DNA are sequences that aid in directing the initiation of transcription to the proper site. In some eukaryotic genes these sequences conform to an initiator (Inr) consensus element. The human β-globin gene has been shown to possess an Inr element that is functional in transcription reactions performed in vitro.

Polyadenylation at the 3′ end of mRNA precursors depends on a signal in the 3′-untranslated region, generally AAUAAA (AATAAA in genomic DNA). The mechanism of modification of the 3′ end is complex and involves not only polyadenylation but also cleavage of the precursor RNA because the primary RNA transcript extends several hundred nucleotides past what becomes the position at which the poly(A) tail is added.

Translation of mRNA into a polypeptide proceeds by the reading of triplets (codons) on cytoplasmic ribosomes. The first AUG codon (specifying methionine) present in the mRNA specifies the start site for translation of the mRNA into protein and is embedded in a sequence context (the Kozak consensus sequence, typically CC[A/G]CC ATG G) that signals the binding of translation initiation factors and ribosomes to the mRNA. Usually the amino-terminal methionine residue is removed from the growing polypeptide chain even before its synthesis is completed. Termination of polypeptide chain translation is directed by the termination codons UAA, UAG, or UGA. Mutation of these codons allows continued translation into the 3′-untranslated sequences of mRNA, as occurs in selected α-globin chain variants associated with α-thalassemia (discussed later).

As briefly reviewed earlier, the formation of functional mature mRNA demands extraordinary precision and depends on highly conserved sequence elements. As exemplified by the thalassemia syndromes, point (or other) mutations in these signals lead to reduced or absent polypeptide chain synthesis, which is the hallmark of thalassemia. Mutations causing thalassemia involve all phases of gene expression, including gene transcription, RNA splicing, integrity of the coding sequence, 3′ polyadenylation, and translation initiation (discussed later).

Proximal Regulatory Sequences and Transcription Factors

Several conserved sequence elements (motifs) in the 5′-flanking sequences of globin genes comprise the promoter, a region required for accurate and efficient transcription of genes by RNA polymerase II. Promoters of vertebrate globin genes are similar in overall configuration and subset of motifs present but differ in their detailed organization and sequences. Promoters generally cooperate with more distant regulatory elements, termed enhancers, to stimulate transcription. As discussed later, globin gene promoters appear to interact in a synergistic fashion with very powerful distant elements known as locus control regions (LCRs).

The TATA (or ATA) box is a motif seen in nearly all promoters, including those of the globin genes. The TATA box, typically located 20 to 30 base pairs (bp) upstream from the transcription start site, constitutes the binding site for a general transcription factor, the TATA-binding protein (TBP). Binding of TBP to the TATA box is the first step in the assembly of a basal transcription complex (often termed TFIID ) that includes many additional proteins (such as TFIIA, TFIIB, TFIIE/F, and TFIIH) and RNA polymerase II. Mutations within the TATA box, as occur in some types of β-thalassemia, decrease the binding of TBP to the promoter and decrease transcription.

DNA sequence motifs located upstream of the TATA box bind proteins that interact with the general transcription machinery through protein-protein contacts with the TFIID complex and other associated proteins. These promoter-bound proteins may either increase (activate) or decrease (repress) the rate of transcription. A small set of motifs are consistently present in globin gene promoters, including the CCAAT box, the CACC box, and GATA consensus sequences. Each motif may be viewed as a potential binding site for one or multiple transcription factors, which are either tissue restricted or ubiquitous in their cellular distribution.

Transcription factors are typically viewed as modular proteins made up of two domains that fulfill different functions: a DNA-binding domain responsible for sequence-specific DNA recognition and an activation (or repression) domain or domains that interact with components of the basal complex to modulate transcription. Additionally, some transcription factors may function to recruit activities that modify the histones around which DNA is wrapped to make chromatin. These modifications can indirectly alter the activity of the basal transcriptional complex. It is currently believed that transcriptional specificity is achieved by functional cooperation and interaction between cell-restricted and general transcription factors. As background for understanding globin gene control, the presently characterized erythroid-enriched transcription factors are reviewed here. For additional discussion of these proteins, readers are referred elsewhere.

The consensus motif (A/T)GATA(A/G), known as the GATA motif, is found in the promoter region of most vertebrate globin genes and binds an abundant erythroid-restricted transcription factor, GATA1. GATA motifs have been identified in the regulatory elements of virtually all erythroid-expressed genes, consistent with the notion that GATA1 should serve a critical role in erythroid gene expression. As noted later, multiple GATA sites are also present within distant regulatory elements. The essential role of GATA1 in erythroid development was formally demonstrated through gene targeting experiments in mouse embryonic stem cells. Disruption of the single X-chromosome GATA1 gene in totipotent embryonic stem cells prevents their development into normal erythroid cells. Naturally occurring but rare hypomorphic mutations of GATA1 in humans have been shown to cause β-thalassemia or disrupt normal erythroid development. The GATA1 protein is a member of a small family of related “GATA factors” that are distinguished by a novel zinc-finger DNA-binding domain. In addition to merely specifying DNA recognition, this domain also mediates protein-protein interactions. Accordingly, GATA1 is able to interact physically with other GATA1 molecules or with other types of zinc-finger proteins, including the ubiquitous CACC- or GC-binding factor Sp1, the transcription factor erythroid Krüppel-like factor (EKLF), and a specific cofactor called FOG-1 (for “friend of GATA1” ; discussed later). It is envisioned that through its multiple physical interactions, GATA1 cooperates with other transcription factors, perhaps bound to DNA at distant sites, to program erythroid-specific transcription.

CACC motifs, which are represented by diverse sequences within globin and other gene promoters, bind a variety of transcription factors. Many CACC sequences are recognized by Sp1, a ubiquitous zinc-finger activator protein. A particular CACC motif, CCACACCCT, is found in the adult β-globin gene promoter and is recognized with high affinity by the erythroid-specific protein EKLF (also known as KLF1). The functional relevance of this binding site has been established through naturally occurring mutations that lead to β-thalassemia (discussed later). In addition, gene targeting (or knockout) experiments in mice have formally established that EKLF/KLF1 is necessary for efficient β-globin transcription in vivo.

A third erythroid transcription factor, known as NF-E2, binds to an extended motif—(T/C)TGCTGA(C/G)TCA(T/C)—that is found within some distant regulatory elements (discussed later) and a small subset of erythroid promoters but not within globin gene promoters. NF-E2 is a heterodimer of two polypeptides of the basic domain–leucine zipper (or “b-zip”) class of transcription factors. One subunit of NF-E2 is tissue restricted, whereas the other is ubiquitous. Although NF-E2 is essential for globin gene expression in mouse erythroleukemia cells in tissue culture, its role in vivo appears to overlap that of one or more unknown factors that may act through the same target sites in DNA.

Locus Control Regions and Chromatin Domains

How is globin gene transcription activated and developmentally controlled? Inspection of the DNA sequences of globin gene promoters in the early 1980s failed to provide substantive insight. Initial attempts to dissect control elements involved introducing globin genes into the germline of mice by oocyte injection but were plagued by low-level and erratic transgene expression. Nonetheless, it was possible to show that low-level stage specificity was imparted by the human β- and γ-globin gene promoters. For example, when the human β-globin promoter is introduced into transgenic mice, it directs gene expression only in adult erythroid cells, whereas the human γ-globin promoter is active only in embryonic erythroid cells (mice do not have an HbF stage).

These early globin gene regulation studies suggested that critical regulatory elements required for high-level expression were missing from the immediate vicinity of the genes themselves. When sensitivity to digestion by the enzyme DNase I was used as an indicator of chromatin structure in the mid 1980s, regions of extreme sensitivity (hypersensitivity sites [HSs]) were identified far upstream (≈30 to 50 kilobases [kb]) of the adult human β-globin gene. Four subregions were delimited; they are present in the chromatin of erythroid, but not in nonerythroid cells. An additional site located even further upstream was found in all tissues. In a formal test of their functional relevance, the HSs were linked to a human β-globin and introduced into the germline of mice. Remarkably, transgenic mice then expressed the human β-globin gene at a level equivalent to that of the endogenous mouse β gene. Further studies showed that the transgene is expressed not only in a tissue-specific manner but also in a copy number–dependent fashion, independent of the chromosomal site of integration. These HSs comprise an essential distal regulatory domain, now referred to as the LCR (see elsewhere for a more detailed history and discussion).

Other globin gene clusters also contain erythroid-specific DNase I HSs. A segment of extreme DNase I hypersensitivity (known as HS-40) located far upstream of the human α-globin genes serves as an enhancer for the α locus. HS-40, however, does not display the full properties of an LCR because it does not direct copy number–dependent transgene expression. Nonetheless, the in vivo relevance of both the β-LCR and HS-40 is underscored by the discovery of patients with thalassemia who have specific deletions in these regions (discussed later).

The human β-LCR, HS-40, and analogous regions studied in other species are composed of cores, each encompassing a DNase I HS. Cores are approximately 200 to 300 bp in length. Remarkably, within the cores three major protein-binding sites are consistently found: GATA, AP-1 (NF-E2), and CACC sequences. The position-independent activity of the β-LCR correlates best with the presence of GATA and CACC motifs, particularly within the subregion known as HS-3. Enhancer activity of the LCR, particularly within subregion HS-2, requires the NF-E2 motif. The protein-binding motifs within the LCR are also found in globin and other erythroid-expressed gene promoters. To date, no protein-binding sites unique to LCR elements have been identified. Hence the distinctive properties of the LCR (or HS-40) appear to reflect the synergistic interactions of more typical transcription factors rather than the action of a new set of regulatory proteins.

The discovery of distant control elements, marked by DNase I hypersensitivity, emphasizes the relationship between chromatin structure and globin gene regulation, an association solidified by the unraveling of a rare syndrome, α-thalassemia with X-linked mental retardation. This condition results from mutations in a gene designated XH2 (or ATRX ) that encodes a member of the helicase superfamily. Such proteins, which are often involved in DNA recombination and repair and in the regulation of transcription in Drosophila , yeast, and mammals, appear to influence transcription in a global manner by altering chromatin structure.

Regulation at a Distance: Globin Gene Switching

How do LCR sequences influence globin gene transcription over large distances (>50 kb), and how are the individual globin genes developmentally regulated? Two formal possibilities have been considered. On one hand, the LCR might merely provide an environment conducive for activation of the downstream globin genes. The globin genes would be autonomously regulated; that is, the developmental profile of their expression would be intrinsic to the individual genes (and presumably determined by their promoters). The “influence” of the LCR is most simply viewed as reflecting physical association of the LCR with globin genes brought into apposition by chromosomal looping. On the other hand, sequential activation of the particular genes might depend (at least in part) on competition of each gene for influence of the LCR such that only one gene-LCR interaction would be productive on a single chromosome at any time. The outcome of the competition would be dependent on the array of proteins bound not only at each promoter but also at specific sites in the LCR. Data in favor of both autonomous and competitive mechanisms of regulation have been obtained (see Orkin for a detailed discussion).

The human embryonic ζ- and ε-globin genes appear to be largely autonomously regulated. Transgenes that contain LCR are expressed during embryonic erythropoiesis (the yolk sac stage) and then extinguished during the fetal liver stage. The information required for shutoff is contained near the globin genes, and competition by adjacent globin genes is not required. Shutoff is hypothesized to reflect the action of repressors, or silencer proteins, that bind the gene promoters. Motifs within the human ε-globin promoter are involved in silencing bind GATA1 and a ubiquitous factor, YY1.

The competitive model of gene regulation is based on experiments in chicken red blood cells that demonstrate competition between the chicken β- and ε-globin gene promoters for a single enhancer located between the genes. In the chicken it has been proposed that an adult stage–specific factor (NF-E4) favors interaction of the β promoter with the enhancer to the exclusion of the ε promoter. In an analogous fashion, data suggest that the human β-globin gene may be negatively regulated in a competitive fashion by the γ-globin gene. Whereas the γ gene is largely autonomously regulated, the β-globin gene is silenced in the embryonic and early fetal stage by a linked γ gene (a γ gene in cis to β). Shutoff of the γ gene, presumably as a result of repressors (or silencers), allows the adult β-globin to be expressed. Although other models are theoretically possible, the capacity of the LCR to act at a distance in regulating activation of globin genes is most compatible with the formation of physical contacts between the LCR (or subregions thereof) and their associated proteins with regulatory elements neighboring the genes themselves. Stage-specific and competitive regulation would therefore reflect engagement of the LCR with genes one at a time. LCR-gene interactions probably have intrinsic stabilities and off-rates such that a single erythroid cell might express more than one globin over time, even from a single chromosome. Experiments examining nascent human globin RNA along the β-gene complex tend to support such models and lend credence to the notion that chromosomal looping brings the LCR and individual genes in apposition. Evidence for LCR interactions with the proximal promoter regions of both the β- and α-globin genes has recently been directly demonstrated by using novel in vivo biochemical approaches. Dynamic interactions between the β-LCR and the γ- and β-globin genes appear to underlie the reciprocal expression of these genes in erythroid cells and provide hope that subtle alterations in the nuclear environment may facilitate reactivation of γ-globin genes in patients with hemoglobinopathies such as sickle cell anemia or β-thalassemia. Recent work suggests that looping of the LCR itself in close proximity to the globin genes may be sufficient to allow for at least partial activation of transcription of these genes, as least in mouse models. This group has recently gone on to demonstrate that such modulation of looping is sufficient for activation of embryonic or fetal hemoglobin genes in mouse models and human cells. This offers the prospect that such approaches could be therapeutically useful.

Recent Insights into Developmental Globin Gene Switching from Human Genetics

The molecular basis of the fetal-to-adult hemoglobin switch had remained an enigma over the course of nearly three decades. Although regulators of globin gene regulation, including GATA1, EKLF/KLF1, and NF-E2, had been identified, none of these regulators appeared to be sufficient to confer the stage-specific expression properties that are observed for the γ- and β-globin genes. By following up on the results of genome-wide association studies (GWAS)—a technique by which associations between common polymorphisms across the genome and various traits are tested—the first developmental stage-specific regulator of the fetal-to-adult hemoglobin switch, BCL11A, was identified (see Fig. 21-3 ). Initial GWAS had identified a robust signal of association on chromosome 2 with HbF levels in both healthy persons and in patients with sickle cell disease. This signal was most robust within a regulatory element of the gene BCL11A , which encodes a multizinc finger transcriptional regulator that had previously been implicated in B lymphocyte and neural development. By examining the hypothesis that BCL11A may also regulate HbF expression, it was found that reducing BCL11A expression allowed robust reactivation of HbF in adult cells. Consistent with this finding, BCL11A is developmentally regulated and its expression is maximal in erythroid cells with little or no γ-globin expression. Moreover, BCL11A occupies regulatory elements within the β-globin locus, although it does not bind the promoter of the γ-globin genes. BCL11A holds tremendous promise as a therapeutic target for HbF induction in patients with β-thalassemia or sickle cell disease. Indeed, a proof of concept study has shown that removal of BCL11A in the hematopoietic system of mouse models of sickle cell disease can result in amelioration of disease in this model system. An erythroid-specific enhancer element of BCL11A has recently been identified and holds promise for tissue-specific targeting of BCL11A expres­sion. Furthermore, recent insight from rare human deletions within the β-globin gene cluster, which will be discussed in more detail later in this chapter, are providing important mechanistic insight into how BCL11A acts to regulate the fetal-to-adult hemoglobin switch. It is likely that further studies of the type described here will lead to targeted approaches by which BCL11A activity can be selectively modulated.

In addition to the insight gained into hemoglobin switching from studies of BCL11A, other work has suggested that rare mutations in KLF1/EKLF may lead to elevations of HbF levels in patients and that this transcription factor may be involved in hemoglobin switching through the regulation of BCL11A (see Fig. 21-3 ). However, other mutations in KLF1 result in either a significant congenital anemia or have little affect upon globin gene regulation, suggesting that other factors may modify the observed phenotypes in the patients. Therefore further study of KLF1 and its role in this regulatory process is needed. In addition to the GWAS highlighting the role of BCL11A in HbF regulation, these studies have also suggested a role for the MYB gene in silencing of HbF. This role has been substantiated through functional studies that have shown that reducing MYB levels can robustly activate γ-globin expression. GWAS of β-thalassemia patient populations have shown that variants upstream of MYB can have a profound affect on the clinical course, and therefore MYB may be an important therapeutic target. However, further studies are needed to better understand the exact mechanism by which MYB regulates globin gene expression (see Fig. 21-3 ).

Mutations Affecting Gene Transcription

Point mutations within promoter sequences recognized by transcription factors tend to reduce the affinity with which these proteins bind and typically leads to reduced gene transcription. Analysis of the promoter for the β-globin gene in patients with β-thalassemia has identified a variety of mutations clustered in the ATA and CACC motifs ( Fig. 21-7 ; see also Table 21-2 ). These mutations are associated with preservation of some β-globin expression and hence are customarily associated with the phenotype of thalassemia intermedia. The C→T substitution at position –101, which results in a particularly mild defect, is associated with the “silent carrier” phenotype in heterozygous carriers. Although the CCAAT box is highly conserved in globin genes, no mutations within this motif have been identified in thalassemia. Rare mutations in transcription factors that result in thalassemia have been detected, and exceedingly rare families have been identified in which a thalassemia mutation is unlinked to the globin clusters (see the section on mutations not linked to the globin gene clusters that alter globin gene expression ).

Point mutations in the β-globin gene promoter. The sequences of conserved motifs within the promoter and their distance from the transcription start site are indicated. A single base substitution at the indicated positions results in β ⁺ -thalassemia.

Mutations of the ATA box presumably reduce binding of TBP and therefore lead to decreased transcription initiation. Substitutions in the CACC motifs decrease the affinity of binding by several transcription factors, including the erythroid-specific factor EKLF/KLF1 and the ubiquitous protein Sp1. Studies showing that mice engineered to lack EKLF experience lethal β-thalassemia at the fetal liver stage have established EKLF as a β-globin activator protein in vivo, and patients with mutations in EKLF have reduced production of the adult β-globin gene. Human β-thalassemias resulting from mutation of a single CACC motif are presumably mild because of the presence of one normal CACC motif within the promoter.

In addition to the protein-binding sites in the promoter, proper transcription depends on sequences surrounding the start site of transcription (known as +1). These sequences often display functional activity in in vitro assays and herald the binding of specific protein complexes to this type of element, termed the Inr . Mild β-thalassemia has been associated with a base substitution (A→C) at +1. This substitution has been shown to impair the β-globin Inr. The proteins that mediate this effect are unknown.

A novel mechanism by which transcription at the α-globin locus can be disrupted has been described and serves as a paradigm for a unique class of mutations that can cause human disease. Higgs and colleagues found a variant single nucleotide change upstream of the α-globin genes that creates a binding site for the transcription factor GATA1, which in turn produces a novel promoter that competes with the endogenous α-globin promoters for interaction with upstream enhancer elements such as HS-40. As a result of this mutation, α-globin gene synthesis is reduced and α-thalassemia results. This mutation is present in approximately 4% of the Melanesian population.

RNA Processing Defects in Thalassemia

The importance of RNA splicing for the formation of functional mRNA cannot be overemphasized. As discussed earlier, removal of introns must be precise to the nucleotide for a continuous, translatable mRNA to be generated from an mRNA precursor. As soon as introns were discovered, it was hypothesized that mutations affecting RNA splicing would probably be involved in the thalassemia syndromes. Apart from its role in constructing a functional mRNA, RNA splicing also appears to be a determinant of mRNA stability and is possibly coupled to RNA transport from the nucleus to the cytoplasm.

Mutations That Alter Splice Junctions or Splice Consensus Sequences

Mutations at the 5′ donor site (GT) or at the 3′ splice acceptor site (AG) abolish proper splicing of the pre-mRNA transcript and result in α ⁰ – or β ⁰ -thalassemia ( Fig. 21-8 ; see also Table 21-2 ). Substitutions at other sites within the splice junction consensus sequence have varied effects; because some correctly spliced RNA is produced, albeit a reduced amount, a β ⁺ -thalassemia phenotype ensues.

Examples of abnormal splicing that result from alterations in the splicing consensus sequences. The three β-globin gene exons are symbolized by blue boxes , the normal splicing pattern is illustrated by lines that project above the exons, and the splice donor and splice acceptor sequences of the first intron are shown. Mutations in the invariant GT dinucleotide of the splice donor site abolish normal splicing of the first intron and result in β ⁰ -thalassemia, whereas mutations elsewhere in the consensus sequence preserve some normal splicing and cause β ⁺ -thalassemia. Changes in the splice donor site are associated with abnormal splicing from three cryptic splice donors (lines that project below the exons); one site is within the first intron and results in the addition of intron sequences to exon 1 (brown boxes) . Similarly, changes in the invariant AG dinucleotide of the splice acceptor sequence are associated with β ⁰ -thalassemia, whereas a mutation in an adjacent nucleotide causes β ⁺ -thalassemia.

Mutations within the splice site or the splice site consensus sequences favor improper processing of the mRNA precursor. These secondary splicing events, which are not seen under normal circumstances, occur at positions that resemble splice site consensus sequences. Splicing at these “cryptic” sites generates aberrantly processed, nonfunctional globin mRNA (see Fig. 21-8 ). Mutations within the β-globin IVS-1 splice donor site activate two cryptic donor sites in exon 1 and a third site in IVS-1, whereas mutation in the IVS-2 splice donor activates a cryptic donor site in IVS-2. Mutation of the IVS-2 splice acceptor site activates an upstream cryptic splice acceptor at position 579 in IVS-2. These incorrectly spliced mRNA molecules sustain either insertions or deletions in the coding region and also shifts in the translational reading frame downstream of the cryptic splice site. The polypeptide synthesized beyond this point bears no resemblance to the globin chain and is often prematurely shortened by a termination codon encountered in the new reading frame.

Mutations within Exons That Create an Alternative Splice Site

RNA from β-thalassemia genes with mutations in the IVS-1 donor splice site may be processed via a cryptic splice donor site GTG GT GAGG in exon 1 (codons 24 through 27). Four independent mutations have been identified that activate this cryptic site in the presence of a normal IVS-1 splice donor site ( Fig. 21-9 ; see also Table 21-2 ). These mutations appear to enhance the ability of the cryptic site to compete with the normal site for binding of the splicing complex. A T→A mutation at codon 24 is “silent” at the translational level, yet approximately 80% of RNA transcripts are spliced at this incorrect site; hence, mild β ⁺ -thalassemia ensues. Two mutations—GAG→AAG in codon 26 and GCC→TCC in codon 27—lead to amino acid replacements that produce the hemoglobin variants HbE and Hb Knossos, respectively, in normally processed mRNA. Because a proportion of transcripts are aberrantly spliced, mild β ⁺ -thalassemia results. An analogous mutation in codon 19 produces β ⁺ -thalassemia with the hemoglobin variant Hb Malay, representing mutations that lead to thalassemic hemoglobinopathies.

Mutations that create an alternate splice donor site in the first exon decrease but do not abolish the occurrence of normal splicing (pattern that projects above the exons) and are associated with splicing from the new site in the first exon (pattern that projects below the exons). Three of these mutations lead to the incorporation of a different amino acid into the β-globin chain derived from the decreased quantity of correctly spliced β-globin messenger ribonucleic acid and generate variant hemoglobins.

Mutations within Introns That Create an Alternate Splice Site

Mutations within β-globin IVS-1 may create a new splice acceptor sequence ( Fig. 21-10 ; see also Table 21-2 ). In the first of this class of mutations to be characterized, a G→A substitution at position 110 (19 nucleotides upstream of the normal intron/exon boundary), the majority of globin mRNA precursors are spliced at this alternate site. Because the incorrectly spliced mRNA contains 19 nucleotides from IVS-1, a shift in the reading frame leads to premature termination of translation. A T→G mutation at position 116 of IVS-1 creates a new acceptor site that is used exclusively, thereby leading to little or no normal β-globin mRNA production and to β ⁰ -thalassemia.

Mutations within introns that create a new splice site. Three mutations in the second intron create a new splice acceptor site (the invariant GT is underlined) and activate the identical cryptic splice donor site located just upstream. Abnormal splicing from these sites (pattern that projects below the exons) leads to the creation of a fourth exon (brown boxes) derived from sequences within the second intron. Two mutations in the first intron create a new splice acceptor site with the conserved AG dinucleotide, and abnormal splicing from this site (pattern that projects below the exons) adds sequences from the first intron to the beginning of the second exon.

Three mutations in IVS-2 create new donor sites and activate an upstream cryptic donor site located 579 nucleotides from the exon 2–IVS-2 boundary (see Table 21-2 and Fig. 21-10 ). The consequence of these mutations is the insertion of a fourth “exon” derived from sequences within IVS-2. Although the normal donor and acceptor sites are unaffected, little or no correctly spliced β-globin mRNA may be produced.

RNA Cleavage and Polyadenylation Defects

Proper cleavage at the 3′ end of the pre-mRNA and subsequent poly(A) addition depend on the integrity of the AAUAAA signal in the 3′-untranslated region. The importance of the polyadenylation signal for efficient production of globin mRNA was first demonstrated in α-thalassemia. An AAUAAA→AAUAAG mutation in the α ₂ gene reduces the efficiency of cleavage-polyadenylation of precursor RNA and leads to “run-on” transcripts that terminate downstream of the gene (see Fig. 21-12 ). Mutations in the AAUAAA element have also been described in β-thalassemia, in which the presence of elongated in vivo transcripts has been demonstrated. The transcripts appear to terminate at the next AAUAAA signal, which is present approximately 900 nucleotides downstream of the normal cleavage site. These mutations lead to a moderate reduction in the level of β-globin mRNA and a β ⁺ phenotype.

Mutations Affecting mRNA Translation Initiation

Translation begins at an AUG codon that usually lies within the consensus sequence (GCC)GCC(A/G)CC AT GG. Substitutions within the AUG codon abolish translation, whereas those in other positions of the consensus often result in less efficient initiation of translation.

Four mutations in α-globin genes alter the consensus sequence and impair translation (see Table 21-2 ). Three of them affect the AUG Inr. No globin polypeptide is produced because the next downstream Inr is in a different reading frame. The fourth α-globin mutation in this class, found on a chromosome in which one α-globin gene was deleted, alters the consensus sequence by the deletion of two bp and reduces mRNA translation to 50% of normal. Two AUG Inr mutations of the β-globin gene have been described, and both are of the β ⁰ type (see Table 21-2 ).

Premature Termination (Nonsense) Mutations

Nucleotide substitutions within the coding region are innocuous if they occur in the third position of a codon and do not alter the amino acid inserted during translation. Substitutions that alter codons from one amino acid to another lead to hemoglobin structural variants. Some substitutions change a triplet coding for an amino acid to a stop codon (UAG, UUA, or UGA). Such chain termination (or nonsense) mutations abort mRNA translation and lead to the synthesis of a truncated polypeptide. Moreover, nonsense mutations also reduce the amount of stable mRNA generated, which is a reflection of coupling between mRNA biogenesis and mRNA translation ( Fig. 21-11 ).

Two of the several thalassemia mutations that destroy gene function by the introduction of a premature translation termination codon in β-globin messenger ribonucleic acid (mRNA). The numbers above the individual codons refer to the position of the encoded amino acid in the β-globin. Replacement of C with T in codon 39 introduces the terminator UAG in β-globin mRNA. Another β ⁰ gene has a deletion of the third nucleotide (C) in codon 41, which results in a shift in the reading frame of the mRNA; the new amino acid sequence is shown above the line. This new reading frame has an in-phase terminator (UGA) in a position corresponding to codon 60 to 61.

(Modified from Nienhuis AW, Anagnou NP, Ley TJ: Advances in thalassemia research. Blood 63:738–758, 1984.)

Chang and Kan described the first nonsense mutation in β-thalassemia in which a lysine codon at amino acid position 17 was converted to a stop codon (AAG→UAG). Although no β-globin chains were produced in vivo, complete translation of the abnormal mRNA could be achieved in a cell-free extract capable of protein synthesis by the addition of a “suppressor” transfer RNA (tRNA) that inserts a serine at the UAG codon. Several other nonsense mutations causing thalassemia have been described ( Fig. 21-12 ; see also Table 21-2 ). In addition, single or dinucleotide insertions or deletions have been observed that alter the translational reading frame and introduce a premature stop codon as a consequence. Two termination mutations have been described in the α-globin genes, one that introduces a stop codon and the other a frameshift. In addition, frameshift mutations have been described that result in abnormal elongation of globin chains (see the section on unstable β-globin chains ).

Ribonucleic acid cleavage and polyadenylation occur 15 to 20 bp downstream from the AAUAAA polyadenylation signal. Mutational analysis in the rabbit β-globin gene has established that sequences located downstream from the polyadenylation site, called the G/U cluster, are also required for efficient cleavage and polyadenylation. Individual point mutations at one of several nucleotides within the AAUAAA polyadenylation signal result in β ⁺ -thalassemia. The same A→G mutation in the last position that causes β-thalassemia has been observed in the α ₂ -globin gene and results in α ⁺ -thalassemia.

mRNA molecules with termination mutations often do not accumulate to a normal level in vivo. The extent of this effect is variable and depends on the specific mutations; deletion of the third nucleotide (C) from codon 41 ( Fig. 21-13 ) leads to complete absence of globin mRNA, whereas a single substitution in the β39 codon allows the accumulation of roughly 5% to 10% of the normal amount of globin mRNA. The basis for the quantitative deficiency in these mRNA species is of considerable interest. Some data suggest that such mutations lead to intranuclear degradation of abnormal globin RNA and suggest a link between mRNA translation and nuclear RNA processing or nuclear to cytoplasmic transport of mRNA. Experimental studies in tissue culture systems have shown that the deficiency in β-globin mRNA accumulation is specific for nonsense mutations and is not observed with missense mutations ; a suppressor tRNA that allows the abnormal mRNA molecule to be translated completely will correct the quantitative deficiency in globin mRNA. Recent work from a number of investigators has begun to unravel the molecular machinery that mediates this phenomenon, which has been termed nonsense-mediated decay , and appears to play an important role in normal physiology, as well as pathologic states.

Point mutations in the terminator codon of the α-globin gene that lead to the synthesis of elongated α-globins. The normal terminator, UAA, of α-globin messenger ribonucleic acid is shown in the center. Each of the nine possible single nucleotide substitutions is depicted; two would result in the formation of another terminator codon, whereas the other seven would lead to insertion of an amino acid at this position and continued synthesis of the globin chain. Four such mutations have been described; the mutation that causes the synthesis of hemoglobin (Hb) Constant Spring (CS) is the most common.

(Modified from Weatherall DJ, Clegg JB: The thalassemia syndromes . Oxford, 1981, Blackwell Scientific, p. 578.)

Termination Codon Mutations

UAA is the normal termination codon for both α- and β-globin mRNA translation. The 3′-untranslated regions are 109 and 132 nucleotides for α- and β-globin mRNA, respectively. A single nucleotide substitution in the termination codon could either create another stop codon (UAG) or permit incorporation of an amino acid at this position and translation of the otherwise untranslated 3′ sequences until the next in-frame stop codon. Four termination codon mutations involving the α ₂ gene have been reported (see Table 21-2 ). These mutants differ only in the specific amino acid incorporated at the terminator codon position (see Fig. 21-13 ). Translation terminates in each instance at a UAA codon in the polyadenylation signal (AA UAA A) downstream, and a 172–amino acid polypeptide is produced. The first of these elongated α chains to be described was found in Hb Constant Spring (CS). The α chain in this hemoglobin has a glycine substituted at codon 142. Hb CS produces an associated thalassemia phenotype because of a marked reduction in α ₂ -globin mRNA stability.

Mutations that give rise to elongated β-globin chains have also been described. Hb Tak is a 157–amino acid product of a β-globin mRNA molecule containing two inserted nucleotides in the terminator codon 147. An analogous elongated β-globin with 157 amino acids found in Hb Cranston reflects a dinucleotide insertion in codon 147, but red blood cells containing Hb Cranston are morphologically normal. The mechanism by which the β ^Tak mutation causes thalassemia has not been elucidated.

Mutations Affecting Globin Chain Stability

Hemoglobin Assembly.

Shortly after synthesis is completed, α- and β-globin chains bind a heme moiety and rapidly associate into α ₁ β ₁ dimers in a noncovalent reaction that is nearly irreversible under physiologic conditions. The majority of the heme contact points are present in the portion of the globin chains encoded by exon 2, whereas most α ₁ β ₁ contacts are located within the exon 3 domain ( Fig. 21-14 ). These dimers may then reversibly associate with other dimers to form the hemoglobin tetramer. Formation of the α ₁ β ₁ dimer, therefore, is the principal controlling step in the assembly of hemoglobin. This subject is discussed in greater depth in Chapter 19 .

Schematic representation of the α- and β-globin chains and the location of residues involved in different hemoglobin functions ( black vertical bars ). Heme contact sites and α ₁ β ₂ contacts are concentrated in the second exon ( blue ), whereas α ₁ β ₁ contacts are principally located in the third exon ( salmon ). DGP, Diphosphoglycerate.

(Modified from Eaton WA: The relationship between coding sequences and function in haemoglobins. Nature 284:183–185, 1980.)

Hemoglobin assembly is an important determinant of the final hemoglobin composition of the erythrocyte. The rate constant of dimer formation depends greatly on the surface electrostatic charge of the subunits. α-Globin has a net positive surface charge, whereas β-globin has a net negative surface charge. The other normal β-like globin chains, γ-globin and δ-globin, dimerize with the α chain at a lower rate. δ-Globin has a lower net negative surface charge than does β-globin, and the significant structural differences between γ- and β-globin presumably account for the differing dimerization rates. When β-globin chains are in limited supply (i.e., β-thalassemia), HbA ₂ and HbF levels may increase because of enhanced dimerization with α chains, independent of changes in the production of δ and γ chains. In α-thalassemia or iron deficiency, HbA ₂ and HbF levels will decrease because of competition with β chains for the limited number of α chains. Similarly, β-chain variants may have decreased (β ^S and β ^E ) or increased (β ^Baltimore ) affinity for α chains based on their net surface charge. The net hemoglobin composition of the cell is determined by these simple rules of competition based on the relative affinity of hemoglobin subunits.

An efficient, energy-dependent protein quality control system is present in erythrocytes that rapidly degrades free globin chains while leaving chains that are incorporated into dimers or tetramers unaffected. Changes in globin chain structure that result from amino acid substitutions, premature chain termination, or chain elongation may slow or block the formation of stable α ₁ β ₁ dimers and lead to rapid degradation of the globin chain. In some instances, as discussed later, mutations may also enhance association of the free globin chain with the cell membrane and thereby promote oxidative damage to the membrane and shortened red blood cell survival.

An erythroid-specific α-hemoglobin–stabilizing protein (AHSP) acts as a chaperone for free α-globin chains. In the absence of this protein, mice exhibit mild hemolytic anemia as a result of α-globin precipitation. When this protein is absent in the presence of a β-thalassemia mutation in mice, a much more severe thalassemic phenotype occurs. However, it is currently unclear whether variation in the structure or expression of this gene in humans causes phenotypic variability in patients with β-thalassemia. One study has suggested that this was not the case in patients with HbE–β-thalassemia in Thailand. Another study has suggested that expression levels of this gene vary in humans and that such levels may correlate with the severity of β-thalassemia. Further studies will need to be conducted to better understand whether AHSP plays a role as a modifier gene in β ⁺ -thalassemia.

During hemoglobin biosynthesis it is also important that the globin chains not be produced in excess of heme. A protein called heme-regulated inhibitor (HRI) kinase appears to mediate this function by inhibiting globin chain translation when an erythroid precursor is deficient in heme. When HRI is absent in mice with a β-thalassemia mutation, the thalassemic phenotype is exacerbated and lethality of the mice results.

Unstable β-Globin Chains

Several unstable β-globin chains have been associated with thalassemia (see Table 21-2 ). Five mutations lead to amino acid substitutions in the β-globin chain. Frameshift mutations in the third exon result in the synthesis of an elongated β-globin chain with a novel carboxy-terminal. A premature termination mutation in exon 3 has also been described. Many of the unstable β-globin chain mutations in exon 3 are associated with a dominantly inherited form of thalassemia (see the section on dominant β-thalassemia ). Thein and colleagues proposed that alterations in β-globin structure in exon 3 interfere with α ₁ β ₁ dimer formation yet may permit binding of heme to the mutant globin chain through contacts in the exon 2 domain. These free, heme-associated β chains may be more resistant to proteolysis and associate with the cell membrane, where they form “inclusion bodies” and induce oxidative damage.

Thalassemic Hemoglobinopathies

The mutations described in this section, taken together with the RNA-processing mutants HbE, Hb Knossos, and Hb Malay and the termination mutant Hb Tak, include a distinctive set characterized by structural changes in the hemoglobin molecule and a thalassemia phenotype. These mutations are often referred to as “thalassemic hemoglobinopathies” (see the section on thalassemic hemoglobinopathies ) and are characterized clinically by a syndrome of ineffective erythropoiesis. Other globin variants may be associated with mild hypochromia, microcytosis, and chronic hemolysis because of instability and degradation of hemoglobin tetramers and are discussed in detail in Chapter 19 . Because thalassemia is the consequence of an imbalance in α- and β-globin chains, these variants are not considered part of the spectrum of thalassemia.

Haplotype Analysis

The first several β-thalassemia mutations were identified by cloning and sequencing β-globin genes isolated from persons with β-thalassemia major. Because certain mutations are extremely common, a nondirected strategy is inefficient because β genes with common mutations will be repeatedly studied. For example, 95% of the β-thalassemia alleles on the island of Sardinia contain the codon 39 nonsense mutation.

To facilitate the search for new β-thalassemia mutations, Orkin, Kazazian, Kan, and others introduced the concept of haplotype analysis to the study of thalassemia. Naturally occurring, genetically neutral, nonselected sequence differences among individuals constitute polymorphisms, which are estimated to occur roughly once every 100 bp. These sequence differences are heritable, and those residing close to one another on a chromosome tend to be inherited together, a property known as linkage . A subset of polymorphisms will alter the cleavage site for a restriction enzyme or create a site where one did not exist. Therefore, when DNA from unrelated individuals is digested with a restriction enzyme and analyzed by Southern blotting, polymorphisms in the restriction enzyme digest pattern may be observed and are referred to as restriction fragment length polymorphisms (RFLPs). Within the 60 kb of the human β-globin cluster, more than such 20 RFLPs are known ; at least 13 have been identified in the α cluster. The pattern of these RFLPs (each based on the presence [+] or absence [−] of a restriction enzyme cutting site) along the chromosome defines a haplotype of associated or linked polymorphisms. Seven RFLPs were used initially to define nine distinct haplotypes (I through IX) of the β-gene cluster in the analysis of thalassemia mutations in Greek and Italian populations from the Mediterranean basin ( Fig. 21-15 ).

Linkage of chromosomal haplotypes to specific β-thalassemia mutations in Mediterranean populations. A haplotype is defined by the sequential pattern of restriction enzyme sites (present “+” or absent “−”) along a chromosome. In this example, seven restriction enzyme sites were used to classify nine haplotypes. Overall frequency refers to the prevalence of the specific haplotype as found in all persons, with or without thalassemia. IVS, Intervening sequence.

(Modified from Orkin SH, Kazazian HH Jr, Antonarkis SE, et al: Linkage of beta thalassaemia mutations and beta-globin gene polymorphisms with DNA polymorphisms in the human beta-globin gene cluster. Nature 296:627–631, 1982.)

Close inspection of these haplotypes revealed a nonrandom association of restriction digest patterns within the β-gene cluster ( Fig. 21-16 ). The pattern of restriction sites 5′ of the δ-globin gene is inherited as a group, whereas restriction sites downstream (including the β-globin gene) track as another set. In all populations only a few haplotypes predominate. The full spectrum of haplotypes is derived from random association over evolutionary time between the 5′ and the 3′ subhaplotypes, presumably reflecting the presence of a recombination “hot spot” lying between these regions.

Restriction endonuclease sites for which restriction fragment length polymorphisms (RFLPs) have been identified in the β-globin gene cluster. A recombinational hot spot has been identified between the two brackets; the RFLP enclosed in each bracket must often remain associated during recombination (see text).

The generation of haplotypes appears to be an ancient event predating racial dispersion. Consequently, a specific haplotype may be found in diverse ethnic and racial groups from different geographic locations. The introduction of malaria as a selective pressure for certain random mutations is a more recent phenomenon. A mutation leading to thalassemia would be under positive selection and amplified within a population; accordingly, the mutation resides on the haplotype background in which it originated in that ethnic group. Several conclusions were derived from the study of different racial groups. Within a single population both normal and thalassemia β-globin genes are found on the same haplotype, but specific thalassemia mutations tend to be linked to a single haplotype. Individual thalassemia mutations are generally restricted to a single population (see Table 21-2 ). In circumstances in which specific mutations are found in different populations, the identical thalassemia mutation may have occurred and been selected independently and will be found on a different haplotype background. This observation has provided a sound genetic basis for the belief that thalassemia has had multiple distinct origins throughout the world. In circumstances in which a specific mutation is found on more than one haplotype within a population, the 3′ subhaplotypes (where the β-globin gene resides) may be identical, whereas the 5′ subhaplotypes differ because of recombination between the two subhaplotypes (for an example, see codon 39 mutation, see Fig. 21-15 ). By this mechanism, specific mutations can be distributed to new haplotypes within an ethnic group, and an independent origin of the mutation need not be invoked.

New thalassemia mutations were identified by cloning thalassemia β-globin genes from distinct haplotypes within a population, thereby avoiding the likelihood of repeated cloning of the same common mutation. In this way, the great diversity of thalassemia mutations was elucidated.

Crossover Globins: Hemoglobin Lepore and Hemoglobin Kenya

Deletion mutations in the α- and β-globin gene clusters arise through unequal homologous recombination or through nonhomologous (illegitimate) recombination. In contrast to the α-globin cluster, in which there are blocks of tandem duplicated sequences (discussed later), the only directly repeated homologous segments of DNA in the β cluster are the globin genes themselves. Hence mutations arising from homologous but unequal crossover in the β-globin cluster are relatively uncommon and usually involve two globin genes directly ( Fig. 21-18 ). Two crossover hemoglobins, Hb Lepore and Hb Kenya, are associated with thalassemia.

Schematic representation of the unequal crossover events that occur during meiosis and result in formation of the Lepore and anti-Lepore ( A ) and the Kenya ( B ) genes.

(Modified from Nienhuis AW, Benz EJ Jr: Regulation of hemoglobin synthesis during the development of the red cell. N Engl J Med 297:1318, 1977.)

In 1958, Gerald and Diamond identified a minor hemoglobin component by starch gel electrophoresis in the blood of parents of a patient with thalassemia major. Structural analysis revealed that the hemoglobin was composed of α-globin and a heretofore undescribed globin consisting of fusion of the 80 to 100 amino-terminal amino acids of δ-globin with the carboxyl portion of β-globin. This “chimeric” globin polypeptide, named Lepore after the family in which it was first described, arose from an unequal, homologous recombination event between the δ-globin and β-globin genes ( Fig. 21-18 ). It is poorly expressed because transcription of the fusion gene is under control of the δ-gene promoter. Other Lepore-type globins have been characterized in which the relative contribution of the δ and β genes to the fusion protein varies as a result of a different point of crossover. Homozygotes for Hb Lepore have 90% HbF and approximately 10% Hb Lepore, but no HbA or HbA ₂ , whereas heterozygotes have mainly HbA, 2% to 4% Hb Lepore, and 3% to 5% HbF. In heterozygotes, red blood cells have a very heterogeneous HbF content. Anti-Lepore globins with the amino-terminal sequence of β-globin and the carboxyl sequence of δ-globin have also been described (see Fig. 21-18 ). Persons with anti-Lepore genes also have two normal δ- and two normal β-globin genes; consequently, their red blood cells lack any stigmata of thalassemia. The anti-Lepore globins are produced in very low amounts, perhaps because of sequences within the large intron of the δ-globin gene that may reduce mRNA production.

Hb Kenya, another important crossover hemoglobin, contains a non–α-globin composed of amino sequences of γ-globin and carboxyl sequences of β-globin (see Fig. 21-18 ). Molecular analysis demonstrated that the ^A γ-globin gene was involved in the crossover. The crossover occurred approximately at the position of amino acid codon 100. Hb Kenya was first observed in an individual who was heterozygous for a β ^S gene. This patient had an increased level of HbF (up to 6%) that was uniformly distributed in all his red blood cells. The HbF was entirely of the ^G γ type. Hb Kenya accounted for 17% to 20% of the total hemoglobin. Persons who are heterozygous for Hb Kenya without HbS are clinically healthy. Approximately 10% Hb Kenya and 6% to 10% HbF of the ^G γ type are found homogenously distributed in their red blood cells, in contrast to the distribution of HbF with the Lepore-type deletion.

Silent Deletions

Deletions that eliminate a single γ-globin gene or the δ-globin gene are unlikely to result in a phenotype that would be detected in hematologic screening, and thus few of these mutations have been detected and described. A clinically silent deletion of the ^A γ-globin gene was identified through extensive molecular screening of chromosomes in Melanesians. A 7.2-kb deletion involving the δ-globin gene but not the γ- or β-globin gene was reported in Corfu (δβ) ⁰ -thalassemia in cis to the G→A substitution at IVS-1 position 5. Homozygotes for the IVS-1 position 5 mutation ordinarily exhibit a mild β+-thalassemia intermedia phenotype with 10% to 20% HbA, but homozygotes for Corfu (δβ) ⁰ -thalassemia display the clinical phenotype of β ⁰ -thalassemia intermedia with 100% HbF. This finding has been interpreted as evidence that sequences in the deleted region are important in activating the β-globin gene and potentially in suppressing γ-gene expression. Subsequently, however, the identical deletion was identified in cis to a normal β-globin gene. In this circumstance, the deletion leads to failure of δ-globin gene expression but has no demonstrable effect on expression of the γ- and β-globin genes on the same chromosome. Recent work has suggested that this deletion may increase γ-gene expression in cis to the deletion but that translation of the γ gene may be limiting in certain settings, thus helping explain the discrepancy between carriers of the deletion and homozygotes. The previously discussed mapping studies of HPFH and δβ-thalassemia deletions suggest that the regulatory element upstream of the δ-globin is important for γ-globin silencing, consistent with the idea that the Corfu deletion is removing this critical regulatory element and therefore can result in elevated HbF levels.

β ⁰ -Thalassemia Deletion Mutations

Several deletions remove the β-globin gene and are associated with typical high-HbA ₂ β ⁰ -thalassemia (see Table 21-4 ). Interestingly, deletions that remove the promoter region are associated with somewhat higher HbF levels than usually seen in β ⁰ -thalassemia heterozygotes (e.g., see Oner and colleagues ). The HbF is distributed in a heterocellular pattern. In contrast, a 0.6-kb deletion at the 3′ end of the β gene that spares the β-globin promoter, seen in Asian Indians, is not associated with an increased level of HbF in heterozygotes. One explanation for the different phenotypes relates to the possibility that an intact β-gene promoter may interact productively with the LCR and thereby effectively compete with an incompletely silenced γ-globin gene at the adult stage. Removal of the promoter may alleviate competition and favor persistent γ-globin expression.

δβ-Thalassemia

Persons heterozygous for these deletions generally have 5% to 20% HbF composed of both ^G γ- and ^A γ-globins and distributed in a heterocellular fashion (see Table 21-4 ). The erythrocytes are typically hypochromic and microcytic. The level of HbA ₂ is characteristically low in comparison with the modest elevations observed in most persons with β-thalassemia trait. Homozygotes for these deletions produce only HbF yet exhibit a mild β ⁰ -thalassemia intermedia phenotype with hemoglobin levels of approximately 10 g/dL. The most common of these deletions is referred to as the Sicilian type. The deletion extends from within the δ-globin gene to just beyond the β-globin gene (see Table 21-4 ). As previously discussed, recent work comparing such deletions with the similar HPFH deletions has provided insight into potential regulatory regions needed for HbF silencing and which are bound by the critical HbF silencing factor, BCL11A.

^A γδβ-Thalassemia

The phenotype of these deletions in heterozygotes is identical to that of δβ-thalassemia: hypochromic microcytic red blood cells with low levels of HbA ₂ and moderately increased ^G γ-HbF levels (5% to 15%) with a heterocellular distribution (see Table 21-4 ). Homozygotes have β ⁰ -thalassemia intermedia with 100% ^G γ-HbF. The 5′ breakpoints in these instances lie between the two γ genes or within the body of the ^A γ gene, thereby resulting in silencing of the ^A γ gene together with the δ and β genes. The 3′ end points of several of these deletions have been precisely mapped. Other deletions are very large and extend well beyond the boundary of the β-gene complex (see Table 21-4 ). One interesting mutation of this type, the Indian form (see Table 21-4 ), has two deletions, one involving the γ gene and the second involving a portion of the δ and β genes and intragenic DNA. The segment of DNA between the two deletions is inverted such that the 5′ ends of the γ and δ genes come to be adjacent but in an inverted orientation.

γδβ-Thalassemia

The diagnosis of γδβ-thalassemia should be considered in newborns with hemolytic disease associated with hypochromic red blood cells. In adults, it is also a cause of normal-HbA ₂ , normal-HbF β-thalassemia trait. The syndrome was first identified in a newborn with microcytic, hemolytic anemia. Heterozygous β-thalassemia associated with normal levels of HbA ₂ and HbF was identified in the father and many relatives. Decreased γ- and ββ-chain synthesis was demonstrated in the infant’s reticulocytes. The anemia was self-limited; as the baby grew older, the hemolytic anemia disappeared and the phenotype of simple heterozygous β-thalassemia developed in the child.

The syndrome of γδβ-thalassemia results from large deletions within the β-globin gene cluster (see Table 21-4 ). They can be divided into two categories: extremely large deletions that remove the entire β-globin gene cluster or all of the structural genes and deletions such as the Hispanic, English, Dutch, and Anglo-Saxon forms in which the structural genes are left intact but the LCR elements are removed. The remaining genes on the chromosome are not expressed and are found in inactive chromatin that is methylated and resistant to nuclease digestion. Study of these deletions has provided the most convincing evidence that the LCR is required in normal erythroid cells for transcription of the β-like globin genes in vivo. Homozygous γδβ-thalassemia is expected to be incompatible with survival and has not been observed.

Hereditary Persistence of Fetal Hemoglobin Deletion Mutations

Several deletions produce the HPFH phenotype of increased levels of HbF in otherwise normal red blood cells ( Table 21-5 ). * Heterozygotes for these deletions can produce greater than 30% HbF containing both ^G γ and ^A γ chains in a pancellular distribution. Differences in the relative percentages of ^G γ and ^A γ chains can be detected between different types of HPFH deletions (see Table 21-4 ). Heterozygotes have normocytic red blood cells. The red blood cells of homozygotes contain HbF exclusively. The higher oxygen affinity of HbF may lead to slightly elevated hemoglobin levels in such individuals. Although homozygotes are clinically healthy, the α-globin to γ-globin chain biosynthetic ratio may be slightly imbalanced, and mild microcytosis and hypochromia may be observed. This finding suggests that γ-chain synthesis occurs at levels below the output of the normal β-globin gene on these chromosomes.

Several hypotheses have been put forward to account for the increased expression and pancellular distribution of HbF in the deletion HPFH syndromes, particularly when compared with the moderate increase and heterocellular distribution in δβ-thalassemia. As previously discussed, recent studies have examined the differences between several deletional forms of HPFH and δβ-thalassemia. These studies have led to the identification of a region upstream of the δ-globin gene that appears to be a critical binding site for BCL11A and several partner proteins. This observation has also been substantiated through the analysis of the patients with deletional forms of β-thalassemia, where variation in the level of HbF expressed appears to be due to the presence or absence of this upstream region. Further studies of this sort are needed to better understand the exact mechanisms by which this region acts to silence the γ-globin genes.

* See references .

Nondeletion Hereditary Persistence of Fetal Hemoglobin Mutations

Persons with nondeletion hereditary persistence of HbF mutations exhibit elevated levels of HbF with normal red blood cell indices and are identified through hemoglobin screening programs or by study of families in which a segregating high-HbF allele modulates the severity of sickle cell disease or β-thalassemia. In many instances, single base changes have been discovered within the promoter region of either the ^G γ- or ^A γ-globin gene. These mutations are postulated to alter the binding of nuclear regulatory proteins and lead to a more favorable interaction of the promoter with the LCR at the adult stage. Alternatively, these mutations may disrupt the binding of repressor molecules that act to silence the expression of the γ-globin gene. Typically, only increased expression of the γ-globin gene in which the mutation is found occurs.

The level of HbF observed in patients with this syndrome varies greatly and ranges from a few percent to 30% in the −175 T→C substitution found in either the ^G γ- or ^A γ-globin gene. Similar to the deletion forms of HPFH, the HbF may be found in either a heterocellular or a pancellular distribution ( Table 21-6 and Fig. 21-19 ). The −158 C→T substitution in the ^G γ-globin gene is associated with a normal level of HbF in otherwise normal heterozygotes but with a high level of HbF in the presence of erythropoietic stress. For example, in Saudi Arabia, patients with sickle cell anemia often have high HbF levels (25% or greater) and mild disease, whereas their parents with sickle cell trait have normal or only slightly elevated levels. Identification of persons with this mutation (or polymorphism) has been aided by the finding that it is linked to a rare subhaplotype. The −158 HPFH substitution also improves the clinical course of β-thalassemia. A Chinese person homozygous for the −29 promoter mutation has transfusion-dependent β-thalassemia, yet black patients homozygous for the same mutation who co-inherit the −158 HPFH mutation in the ^G γ promoter have mild β-thalassemia.

A , Immunofluorescent staining of erythrocytes with antibody directed against fetal hemoglobin (HbF) shows the distribution of HbF in a heterocellular pattern. B , Immunofluorescent staining of erythrocytes with HbF distributed in a pancellular pattern.

(Courtesy Dr. George Stamatoyannopoulos.)

Confirmation that the base substitutions in the promoter region are the cause of increased γ-chain synthesis rather than associated random DNA polymorphisms is based on several lines of evidence (reviewed by Ottolenghi and colleagues ). As mentioned earlier, they are generally associated with overexpression of the gene in which the mutation is found and typically represent the only sequence change. None of the mutations listed, with the exception of the −158 mutation, have been observed in persons with normal HbF levels. Haplotype analysis in some pedigrees of patients with HPFH has demonstrated that the HPFH determinant is linked to the β-globin cluster. The British type of nondeletion HPFH is quite informative in this regard. The gene has been tracked through three generations, and three homozygous individuals have been observed. Haplotype analysis using restriction enzyme polymorphisms has established linkage of the British HPFH phenotype to the β-globin gene locus. More than 90% of the γ chains are of the ^G γ type, and there is an associated mutation at −198 of the ^G γ gene. Even the three homozygotes, with approximately 20% HbF, have heterogeneous distribution of HbF among their red blood cells. Two homozygotes for −117 ^A γ HPFH have also been described, and they had 24% HbF in a pancellular pattern. More recent transgenic experiments provide support for the idea that the single base substitutions seen in HPFH can lead to enhanced γ-globin expression in adult life.

An interesting aspect of this syndrome is a balanced α-globin to non–α-globin chain synthetic ratio, even in homozygotes, which implies that increased γ-globin synthesis is offset by decreased β-globin synthesis. Expression of the β gene in cis to the mutation may be reduced by 20% to 30%. It has also been observed that HbA ₂ levels are uniformly low in the nondeletion HPFH syndromes and generally correlate inversely with HbF levels. The reduction in δ- and β-globin gene expression in cis to the HPFH mutations is compatible with models of competitive regulation of β-globin expression by the linked γ-globin gene.

Mutations That Remove One α-Globin Gene

The two α-globin genes are embedded in highly homologous, tandem repeated sequence blocks (called X, Y, and Z) that are separated by nonhomologous segments ( Fig. 21-20 ). Unequal, homologous recombination through the X and Z blocks generates a chromosome with a single α-globin gene and another with three α-globin genes. Homologous recombination in the small Y box has not been observed. The most common type of deletion in this class removes 3.7 kb as a result of misalignment of the Z boxes and is known as “rightward deletion.” The products of this crossover are the (−α ^3.7 /) and (ααα ^anti-3.7 /) haplotypes. The −α ^3.7 products may be further subdivided into types I, II, and III by the precise location of recombination within the Z box. Unequal crossover events through the X box leads to “leftward deletion” of 4.2 kb of DNA and the −α ^4.2 chromosome and its triplicated antitype. The incidence of the observed recombination products appears to reflect the size of the homologous target sequence within the boxes because −α ^3.7I (1436 bp) is the most common, followed by −α ^4.2 (1339 bp), −α ^3.7II (171 bp), and −α ^3.7III (46 bp). Unequal α-gene recombination through the X and Z boxes has been reproduced in both prokaryotic and eukaryotic experimental systems with episomal vectors.

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