Version May 2024
INTRODUCTION — The deficiencies in hemoglobin synthesis that characterize the various forms of thalassemia arise from pathogenic variants in or near the two globin gene clusters that encode the globin polypeptide subunits of hemoglobin. To date, no forms of thalassemia arising primarily from defects in iron or heme metabolism have been described. All known forms are due to inherited variants affecting the biosynthesis or post-translational stability of the globin subunits themselves.
Because of their small size, their arrangement into relatively compact gene clusters, and their well characterized physiology, pathology, and clinical genetics, the globin genes were the first human genes to be cloned and thoroughly characterized. The variants that cause the thalassemia syndromes were the first to be defined at the level of their DNA structure. Indeed, the delineation of the impact of these variants on mRNA and protein expression provided the paradigm for the identification of variants in other genes causing other diseases.
The molecular pathology of the thalassemic syndromes will be discussed here. This subject is best understood from the perspective of the normal structure and function of the genes encoding the individual globins [1,2]. The first section of this review will outline the essential features of normal hemoglobin biosynthesis and its underlying molecular biology. (See "Basic genetics concepts: DNA regulation and gene expression" and "Structure and function of normal hemoglobins".)
The second section will discuss illustrative examples of the over 100 different variants causing thalassemia and explain their effects on globin synthesis. The pathophysiologic and clinical aspects of the thalassemic syndromes are discussed separately. (See "Pathophysiology of thalassemia" and "Diagnosis of thalassemia (adults and children)".)
CLINICAL RELEVANCE OF THE MOLECULAR BASIS OF THALASSEMIA — The globin gene clusters and their RNA and globin protein products are the most thoroughly understood in the human genome at the level of their basic structure and arrangement; regulated expression during embryonic, fetal, and adult life; structure-function relationships of the globin proteins; and physiology and pathophysiology of conditions arising from alterations in the genes or elements that control them.
The alpha and beta globin gene clusters are small and compact, such that molecular analysis at the sequence level is readily performed. Thus, any clinician confronted with findings suggestive of a hemoglobinopathy should consult with their clinical pathology laboratory about getting direct analysis of the hemoglobin profile and/or globin gene profile that might aid diagnosis. Very few laboratories possess this capability locally, but nearly all maintain a connection to a reference laboratory. In this author's experience, these reference laboratories also do a good job of guiding interpretation of the results. (See "Methods for hemoglobin analysis and hemoglobinopathy testing".)
GLOBIN GENE ANATOMY AND PHYSIOLOGY
Regulation of globin gene expression — Genes coding for the individual human globins are located in two clusters (figure 1):
●The "beta gene" cluster, located on the terminal portion of the short arm of chromosome 11 (p15), is approximately 70 kilobases long. It contains the embryonic (epsilon), fetal (Agamma and Ggamma), and adult (delta and beta) globin genes coding for the "beta" (or non-alpha) subunit components.
●The 40 kb "alpha gene" cluster, located on the short arm of chromosome 16, contains the embryonic (zeta) and two copies (alpha-2 and alpha-1) of the fetal/adult gene encoding the "alpha" component. While the alpha globins produced by these two genes are identical, the alpha-2 gene is expressed more efficiently than the alpha-1 gene, so that abnormalities of the alpha-2 gene are more apt to be clinically apparent.
Within each gene cluster, the structural genes are separated on both their 3' and 5' ends by variable stretches of non-coding DNA containing several types of regulatory sequences:
●Promoter elements are essential for the binding of messenger RNA polymerase and the initiation of transcription.
●Enhancer and silencer elements stimulate or repress transcriptional activity, depending on the array of transcription factors to which they are bound.
●Stage selector elements facilitate switching from embryonic to fetal to adult hemoglobin; they may represent specialized enhancer and silencer sequences.
●The locus control region (LCR) functions as a "master switch." Both globin gene clusters possess a LCR, located many kilobases upstream of the structural loci (figure 1). The LCR appears to interact with a combination of transcription factors at the onset of erythroid maturation in such a way as to enhance access of the transcriptional machinery and other transcriptional factors to the promoters, enhancers, and silencers within the gene complex. In this sense, some regard the LCR as a "super enhancer." LCR function is absolutely required for expression of globin genes at the extraordinarily high levels needed to support normal hemoglobin production.
Normal hemoglobin biosynthesis requires an intact structural gene and the structural and spatial integrity of silencers, enhancers, promoters, and LCR sequences. Despite considerable effort, we do not fully understand how the sequences and factors regulating globin gene expression interact with one another to ensure high levels of expression of the proper globins at the proper developmental stage and during the proper steps of erythroid differentiation.
However, it does seem clear that the overall activation of the globin genes depends on the mutual and simultaneous binding of key transcription factor and cofactor complexes to cognate enhancer elements in the LCR and enhancer elements adjacent to (or between) the globin genes. This causes formation of a "looped out" chromatin structure that facilitates transcription within the gene cluster. The selection of which genes are expressed at each developmental stage depends on local interactions within the cluster, such as favoring gamma globin transcription and repressing beta globin transcription during gestation, causing Hb F to predominate; the converse occurs during postnatal life, causing Hb A to predominate [3].
Improved understanding of the genomic and epigenomic events surrounding globin gene activation in early erythroblasts has led to the identification of key transcriptional regulator proteins that modulate the patterns of globin gene expression on the basis of their interaction with enhancer or silencer elements in the gene cluster [3]. Of particular relevance to the molecular therapeutics of thalassemia is BCL11A, a transcription factor that is important for activation of immune functions and that in erythroblasts binds near the gamma globin locus and has the net effect of shutting down gamma globin synthesis in postnatal life [4]. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Hemoglobin switching and downregulation of Hb F expression'.)
In animal models, blockade of BCL11A leads to high level of fetal hemoglobin (Hb F) production. Since partial persistence of Hb F correlates with milder disease in beta chain hemoglobinopathies, therapeutic inhibition of BCL11A offers an appealing therapeutic target. Gene therapy trials employing transfer of genes expressing inhibitory RNAs that impede BCL11A production specifically in erythroblasts are showing early promise. (See "Management of thalassemia", section on 'Gene therapy and other stem cell modifications'.)
Non-globin locus variants — Nearly all of the pathogenic variants causing thalassemia reside in or near the globin locus, but at least two interesting exceptions have been reported with the GATA1 and ATRX genes.
GATA1 variants and beta thalassemia — GATA1 is a transcription factor on the X chromosome that is important for activating many genes during erythropoiesis, including globin genes [5]. In individuals with the R216Q point mutation, beta thalassemia occurs in conjunction with thrombocytopenia and dyserythropoietic anemia [6,7]. The basis for selective impairment of the beta locus in the two involved families is unclear but may be related to the impact of the mutation on an interacting factor called "friend of GATA1" (FOG1, also called zinc finger protein multitype 1 [ZFPM1]), or other as yet unknown factors that affect the activation of the beta globin gene disproportionately. (See "Regulation of erythropoiesis", section on 'GATA1'.)
ATRX variants and alpha thalassemia — Pathogenic variants affecting the ATRX gene on the X chromosome are associated with the alpha thalassemia/mental retardation syndrome [8]. The protein product of this gene affects broad patterns of gene transcription and participates in epigenetic remodeling of chromatin.
Somatically-acquired variants at this gene locus appear to account for at least some of the cases of acquired alpha thalassemia (also called acquired Hb H disease) encountered in a subset of patients with myelodysplastic syndromes and more rarely in acute myeloid leukemia (AML) and myeloproliferative disorders [9,10]. Of interest, most of those affected have been older males [11]. (See "Clinical manifestations, diagnosis, and classification of myelodysplastic syndromes (MDS)", section on 'Acquired hemoglobin H disease'.)
Transcription into messenger RNA — The alpha-like genes are 1100 bases long. The beta-like genes are approximately 1800 bases long. Each contains three exons, ultimately coding for globin messenger RNA (mRNA). The exons are separated by two introns, which are transcribed into the mRNA precursor but subsequently excised by pre-mRNA splicing. Splicing knits the exons together in precise register, producing the functional mRNA.
Intranuclear processing of globin mRNA includes the addition of a "cap" structure at the 5' end of the mRNA molecule, and a stretch of adenylate (A) residues (the "poly-A tail") at the 3' end. The 5' cap facilitates efficient translation of the mRNA nucleotide sequence into the amino acid sequence of the globin polypeptide, while the poly-A tail promotes mRNA stability.
The fully spliced and modified globin mRNA is transported from nucleus to cytoplasm. Once regarded as a passive process of diffusion, mRNA transport is now known to be a complex, energy consuming, and, probably regulated process. No forms of thalassemia due to primary defects in mRNA transport have been described. However, impaired mRNA transport may be a secondary abnormality that contributes to reduced accumulation of certain thalassemic globin mRNAs with impaired splicing or translational capacity.
The amount of protein arising from the activity of a globin gene depends on four interacting cytoplasmic processes:
●The amount of mature mRNA delivered to the cytoplasm
●The rate of mRNA translation into proteins by polyribosomes
●The rate at which mRNA is degraded
●The posttranslational stability of the protein
In the case of normal human globin genes, virtually all of the initial pre-mRNA transcripts are delivered in fully functional spliced form to the cytoplasm as mature globin mRNA. The critical need for an equal balance of alpha and non-alpha globin synthesis appears to be met largely by the inherent maximal activity of the alpha and non-alpha genes expressed at each stage of ontogeny. This appears to be assured by nearly equal rates of alpha and beta-like mRNA transcription. However, as will be discussed below, equal synthesis of alpha and beta globin protein depends on the interplay between the individual translational efficiencies and stabilities of alpha and beta-like mRNAs.
mRNA stability — Normal globin mRNAs are extraordinarily long lived. While the typical half-life of most mammalian mRNAs is less than six hours, alpha globin mRNA has a half-life of approximately 30 hours, while that of beta globin mRNA is 15 to 20 hours. Delta globin mRNA is much shorter lived, with a half-life of 3.5 hours, a feature that probably explains why the anucleate circulating reticulocytes, which retain only mRNA synthesized one to two days earlier at the erythroblast stage, produce no new delta globin chains. Gamma, epsilon, and zeta globin mRNAs appear to have stabilities approximately the same, or slightly less, than that of beta globin mRNA.
The extraordinary stability of globin messenger RNAs is due to stabilizing sequences located in the 3' untranslated region (3' UTR) of the mRNA molecule. These stabilizer elements, best characterized for human alpha globin mRNA, bind to proteins that confer resistance to nucleolytic cleavage. Forms of thalassemia resulting from pathogenic variants in these sequences have not been described. However, certain forms of alpha thalassemia, of which hemoglobin Hb Constant Spring is the prototype, exhibit markedly reduced stability, due to secondary disruption of the interactions between 3' UTR stabilizer sequences and their binding proteins. (See 'Failed translation termination: Hb constant spring' below.)
Translation into globin protein — Translation initiation is the process by which the ribosome attaches itself near the 5' end of the mRNA molecule and aligns its translational machinery in a proper reading frame so that the mRNA is translated into the intended sequence of amino acids. Messenger RNA translation is an extraordinarily complex process. The rate limiting step for translation of globin mRNAs is "initiation"; normal translation requires an initiator methionine codon, a continuously open "reading frame" (ORF) in which each three base codon read in succession downstream of the initiator codon codes for an amino acid, and a normally positioned translation termination codon or "nonsense" codon (UAA, UAG, UGA) which codes for no amino acid but specifies the place at which translation should stop.
The initiator codon (AUG) is the essential signal required to mark, for the ribosome, the precise base at which translation of succeeding triplet codons in the open reading frame should commence. Translation will stop when a nonsense codon is reached. Variants convert "sense" codons to "nonsense" codons will result in production of a truncated globin peptide that may or may not accumulate as a stable protein product.
●The beta-like globin polypeptide is 146 amino acids long, not including the initiator methionine, which is cleaved during the translation; the opening reading frame must thus have 147 consecutive "sense" codons, or 441 bases in the ORF. Beta-like globin mRNAs are translated more efficiently than alpha-like globin mRNAs. This appears to reflect differences in the 5' untranslated regions (5' UTRs) of the individual mRNAs, since, if the 5' UTRs of alpha and beta globin mRNA are interchanged, their translational efficiencies change accordingly.
●Alpha-like globins are 141 amino acids long, requiring an ORF of 142 codons, or 426 bases. Normal erythroid progenitors contain slightly more alpha-like globin mRNA than beta-like globin mRNA. This probably reflects the slightly longer half-lives of alpha-like globin mRNAs (see 'mRNA stability' above), and, possibly, a slight excess of alpha globin mRNA transcription.
These differences in translation and mRNA amount appear to balance each other, so that virtually equal amounts of alpha-like and beta-like globin are synthesized in normal erythroid cells.
Hemoglobin assembly — The final steps required for normal hemoglobin production are the post-translational assembly of the four globin chains with four heme molecules and their interaction with one another to form functioning hemoglobin tetramers and ensure their continued stability in the cell.
This process may be facilitated by the presence of two proteins, alpha-hemoglobin stabilizing protein (AHSP), and heme-regulated eukaryotic translational initiation factor 2 (eIF2alpha) kinase (HRI) [12,13]:
●AHSP – AHSP is a chaperone protein that forms a reversible complex with free alpha globin chains [14], preventing the formation of free (excess) alpha chains, inhibiting the production of cytotoxic reactive oxygen species, and stabilizing nascent alpha chains for hemoglobin assembly [15-19]. The physiologically relevant replacement of the chaperone AHSP protein by beta globin chains then leads to the production of the alpha-beta dimer [20], two of which combine to produce the final hemoglobin tetramer, most likely non-enzymatically. Loss, decreased levels, or polymorphisms of AHSP may exacerbate beta thalassemia, a condition in which alpha chains are made in excess [16,21,22].
●HRI – HRI prevents the accumulation of excess alpha- and beta-globin in the absence of heme. Its presence has been shown to reduce the severity of beta thalassemia in mice; the disorder is embryonic lethal in its absence [23].
Experiments in a murine model of beta thalassemia have also suggested that excess alpha chains may be removed by protein quality-control pathways such as ubiquitin-mediated proteolysis and autophagy [24].
The resulting hemoglobin tetramers are extraordinarily stable; nearly all of hemoglobin formed during erythropoiesis remains intact in the red cell throughout its 120-day circulating life span. Some forms of thalassemia arise from mutations that disrupt the formation of the intact tetramer or its stability after formation.
MOLECULAR LESIONS CAUSING THALASSEMIA — Thalassemias result when pathogenic variants in globin genes cause selective deficiencies in the synthesis of alpha-like or beta-like globin. However, it is the imbalance in accumulation of globin subunits that leads to formation of inclusion bodies from unpaired globin chains and the consequent pathophysiology of disease (see "Pathophysiology of thalassemia"). The discussion that follows considers only alpha and beta thalassemias in detail; delta, gamma, epsilon, and zeta thalassemias are rare and usually not associated with significant disease in adults. A few instructive exceptions will be included.
Several hundred pathogenic variants that cause thalassemia have been described [25]. These affect every step in globin gene expression, including the presence and spatial arrangement of the globin gene complex, transcription, pre-mRNA splicing, mRNA translation and stability, and post-translational assembly and stability of globin polypeptides. Only a few examples illustrating each of the major categories will be included here.
Alpha and beta thalassemias will be considered together, because the genetic and molecular mechanisms are quite similar. The distinct features of these syndromes arise from the subsequent behaviors and amounts of the particular unpaired chains accumulating in each instance. (See "Pathophysiology of thalassemia".)
Although there are exceptions, point mutations are the most common causes of beta thalassemia, while deletion of one or more of the four alleles present in a normal diploid erythroid progenitor is the most common cause of alpha thalassemia.
GENE DELETIONS — Deletion of one or more alpha globin genes is the most common mechanism accounting for alpha thalassemia in individuals from Asian countries and Mediterranean regions. On the other hand, complete deletion of the beta globin gene has only been reported rarely, usually as part of larger gene rearrangement lesions. Partial beta globin gene deletions and interstitial deletions have been shown to account for rare cases of beta thalassemia.
Large deletions within the gene clusters
Hereditary persistence of fetal hemoglobin — The "classical" clinically harmless pancellular forms of hereditary persistence of fetal hemoglobin (HPFH) appear to arise from deletional events that remove large regions of DNA from the beta gene cluster. In these cases, 50 to 100 kilobase long segments of DNA are deleted from downstream regions of the beta globin gene cluster. These deletions eliminate the delta and beta globin structural genes.
Persistence of very high levels of fetal hemoglobin (Hb F) synthesis in adult life appears to occur in those cases in which the gene deletion brings a highly active enhancer element into close apposition with the remaining gamma globin gene(s). This enhancer, usually insulated from the globin gene cluster, provides for high levels of gamma globin gene expression and persistence of Hb F into adult life.
Delta-beta thalassemia — Other deletions involving this region cause mild-to-moderate forms of thalassemia in which both delta and beta globin synthesis are absent (delta-beta thalassemia). The clinical prototype seems to depend on the size and location of the downstream deletions; each brings new DNA, with varying enhancer effects, in close apposition to the remaining gamma genes.
Deletions of the LCR — Very rare, but extremely informative forms of both beta and alpha thalassemia have been shown to arise from gene deletion events that remove the locus control region (LCR) sequences [26]. The prototypical mutation, reported in a kindred from the Netherlands, was associated with total absence of beta globin synthesis, even though the beta globin gene and its surrounding promoters and enhancers were normal [27].
This form of thalassemia was found to be due to a large deletion many thousand bases upstream of the structural genes, through which the critical LCR sequences were lost (figure 1). Discovery of this kindred was a major finding that helped to substantiate the critical role played by the LCR in permitting expression of the globin genes during erythropoiesis. A few additional kindreds have been found to inherit thalassemia by a similar mechanism [28].
GENE REARRANGEMENTS — Gene rearrangement events produce several distinctive and informative forms of thalassemia (figure 2).
Hb Lepore — Hb Lepore arises from an unequal crossing over and recombination event between the adjacent, very closely linked (5400 bases apart), and highly homologous delta and beta globin genes (figure 1). These events fuse the 5' end of the delta globin gene (coding for the amino terminus of the delta globin chain) with the 3' end of the beta globin gene (coding for the carboxy terminal region of beta globin). The result is production of a fused (delta-beta) globin polypeptide, containing delta globin amino acids in its amino terminal portion and beta globin amino acids in its carboxy terminal portion (figure 2).
The fused globin (delta-beta) appears to form a functional and stable hemoglobin (alpha(2)/[delta-beta](2)) with altered electrophoretic mobility, called Hb Lepore. Because the production of the abnormal delta-beta globin is under the control of the delta globin promoter, which is only 2 to 3 percent as active as the beta globin promoter, there is severe underproduction of this abnormal globin chain (only 3 to 15 percent of normal) [29]. The chromosomal rearrangement eliminates any intact beta or delta locus from the chromosome, such that the recombination event precludes production of any normal beta globin from that chromosome. Unbalanced production of alpha and beta-like globin results, producing thalassemia of moderate-to-high severity in homozygotes. This form of thalassemia is one of the unusual forms associated with generation of a structurally abnormal hemoglobin variant.
Hb anti-Lepore — Hb anti-Lepore results from the reciprocal chromosome product of the unequal crossover events in Hb Lepore. (See 'Hb Lepore' above.)
The anti-Lepore chromosome acquires a fused (beta-delta) chain, in which the amino terminal region is encoded by beta globin gene sequences and the carboxy terminal by delta globin gene sequences. This hemoglobin is produced at much higher levels, since it is under the control of the beta globin promoter. This chromosome also retains a normal beta and delta globin locus so that no thalassemia results.
Hb Kenya — Hb Kenya is a clinically silent hemoglobinopathy that results from an unequal crossover and recombination event between the beta and A(gamma) globin genes (figure 1) [30]. This crossover produces a fused (A(gamma)-beta) gene. It also eliminates from the recombined chromosome all of the non-coding DNA between the A(gamma) and beta genes, as well as the delta globin gene. The only globin that can be encoded by this chromosome is thus fetal Hb containing only the G(gamma) form, and Hb Kenya containing the fused (A(gamma)-beta) chain (figure 2).
This event is associated with biochemical and clinical features of hereditary persistence of fetal hemoglobin (HPFH), with the added presence of the structural variant, Hb Kenya. The persistence of high levels of fetal hemoglobin synthesis into adult life may arise because of the elimination of stage selector elements and silencers normally located between the gamma and delta genes, and the closer apposition of a strong beta globin gene enhancer normally located at the 3' side of the beta gene to the G(gamma) and Kenya genes. Hb Kenya is rare; the anti-Kenya state has not yet been identified.
Alpha thalassemia — Since each of the two copies of the normal alpha globin genes codes for exactly the same amino acid sequence, it is not possible to create structural alpha globin protein variants analogous to Hb Lepore and Hb Kenya by unequal crossover and recombination. However, direct analysis of globin gene DNA sequences suggests that fairly common forms of alpha thalassemia that appear to arise from a "deletion" of one copy of an alpha globin gene are actually due to unequal crossover and recombination events that fuse the two alpha globin genes together into one.
The reciprocal product, containing three alpha genes, has been found in some kindreds. These excess alpha genes can aggravate the severity of beta thalassemia because of an even greater overproduction of alpha globin chains (table 1) [31].
VARIANTS AFFECTING TRANSCRIPTION — Forms of both alpha and beta thalassemia have been found to arise from pathogenic variants that alter known promoter or enhancer sequences for the alpha or beta globin genes [32]. Some of these are point mutations that alter the efficiency of the promoter or enhancer, while others are small gene deletions or rearrangements that disrupt their spatial integrity. In the aggregate these variants account for only a small percentage of the worldwide burden of thalassemia.
VARIANTS AFFECTING PRE-mRNA SPLICING — Many variants have been described that disrupt normal splicing of the mRNA precursor. Included among these are some of the most common forms of beta thalassemia and some of the more common varieties of nondeletional forms of alpha thalassemia.
Alteration of canonical splice signals — Some thalassemic splicing mutations directly disrupt the canonical "splicing signals" used to mark the beginning and end of each intron so that normal splicing can occur. These short sequences are absolutely required by the splicing apparatus. They signify the places at which excision of the intron and ligation together of the flanking exons should occur.
Certain bases in these splicing signals are absolutely invariant, such as the GT dinucleotide required at the 5' beginning of the intron and the AG dinucleotide required at the 3' end of the intron. The several bases to either side of these invariant nucleotides are consensus sequences within which alteration of the base will change the efficiency with which the site is used. Thus, variations altering these nucleotides can abolish normal splicing or reduce it to a variable degree. Variants that alter the consensus splice sites reduce production of alpha or beta globin mRNA; the pre-mRNA molecules that are not properly spliced appear to be catabolized rapidly, so that no abnormal mRNA products accumulate.
In a few cases, pre-mRNAs that are not spliced at the proper sites are spliced elsewhere by activation of "cryptic" sites, resulting in the production of structurally abnormal, usually non-functioning mRNAs. The molecular basis for variability with which variants reduce the efficiency of normal splicing or generate production of detectable abnormally spliced products remains poorly understood.
Activation of cryptic splicing sites — Another class of splicing variants includes those in which the mutation activates a "cryptic" splice site. One of these variants produces a very common form of thalassemia in the Mediterranean basin. The sites of these variants are not located within the consensus sites at either end of introns. Rather, base substitution, small deletions, or small insertions of DNA, can convert a site within an exon or intron that normally bears only a slight resemblance to a splice site into one containing much stronger consensus signal.
Depending upon the resulting strength of the signals, the splicing apparatus will utilize that site instead of normal site in a greater or lesser percentage of the pre-mRNA molecules being spliced. At least two spliced mRNA products result, accumulating in varying percentages, depending on the efficiency with which the cryptic site is used and the stability of the abnormally spliced product.
This mechanism is best illustrated by a form of thalassemia commonly found in Cyprus [33]. A base substitution converts a cryptic site within the first intron of the beta globin gene into a site that is recognized as a "strong" splice site by the processing apparatus. Ninety percent of the pre-mRNA molecules transcribed from the variant gene are spliced abnormally at the activated cryptic site instead of the normal site. The mRNA product that results retains a small region (17 bases) of intron. These bases alter the reading frame or register for translation, resulting in premature translation termination. The mRNA product is useless for production of beta globin, even though it is sufficiently stable to be detected in the steady state mRNA of reticulocytes. Beta globin can be produced only from the small fraction (10 percent) of the mRNA molecules that are spliced at the normal position. Severe thalassemia results.
Hb E: A special case — Hb E is the prototype of several rare forms of thalassemia in which abnormal splicing results both in reduced accumulation of translatable mRNA and the production of a structurally abnormal globin polypeptide. (See 'Activation of cryptic splicing sites' above.)
Formation of Hb E is due to a single base substitution that alters the coding property of codon 26 in the beta globin gene. As a result, lysine is inserted as the 26th amino acid instead of the normal glutamic acid. This globin forms an (a(2)/beta(2)E) hemoglobin with an altered mobility, called Hb E. (See "Hemoglobin variants including Hb C, Hb D, and Hb E", section on 'Hb E'.)
In some regions of Laos, Cambodia, Vietnam, and southern China, Hb E is present in 15 to 30 percent of the population. Although Hb E exhibits some measurable abnormalities in thermal stability and other properties, it appears to function quite well physiologically when inherited in a simple heterozygous (Hb E trait) or homozygous state, (Hb E disease). These patients have mild-to-moderate shortening of red cell life span and mild symptoms.
However, an individual who inherits Hb E from one parent and beta thalassemia from the other (Hb E-beta thalassemia) has thalassemia, sometimes transfusion-dependent(table 1). The interaction of Hb E with beta thalassemia trait to produce a more severe form of thalassemia was puzzling until the molecular basis for this disorder was deciphered.
The severity of Hb E-beta thalassemia flows from the multiple consequences of the single base change in codon 26. This base substitution activates a cryptic splice site that is sufficiently strong that approximately 65 percent of the pre-mRNA is spliced at that site. The abnormally spliced mRNA is not translatable, highly unstable, and does not accumulate in the cells. The remaining 35 to 40 percent of the pre-mRNA molecules are spliced at the normal site, producing a functional, translatable, and normally stable mRNA. However, it bears the base substitution at codon 26, resulting in translation of the structural protein variant, beta(E) globin, but at only 30 to 40 percent of the normal rate. The gene is thus mildly thalassemic because a stable beta globin is produced at a reduced rate.
This mechanism explains the long-standing observations that Hb E trait presents clinically as a mild form of beta thalassemia trait. Hb E homozygotes have a more clinically similar presentation to patients with more marked, but benign thalassemia trait, while patients inheriting a combination of Hb E and beta thalassemia develop a severe disease. This may also explain why Hb E is such a common variant. It is a thalassemic allele in the malaria-rich regions of the world where other thalassemia alleles are also highly prevalent. (See "Protection against malaria by variants in red blood cell (RBC) genes", section on 'Thalassemia'.)
Several other forms of both alpha and beta thalassemia have been shown to result from molecular mechanisms analogous to that described for Hb E. In most of these cases (eg, Hb Knossos [34]), a structurally abnormal hemoglobin variant is produced in reduced amounts because the majority of the pre-mRNA molecules are misspliced at an activated cryptic splice site created by the same base change encoding the altered amino acid in the structural variant. In a few cases, the globin produced has a normal amino acid sequence because the base substitution is translationally silent. A base substitution in codon 24 that does not alter amino acid coding but does activate a cryptic splice site results in a common, mild form of thalassemia in individuals from African regions [35].
ALTERED mRNA TRANSLATION AND STABILITY — Pathogenic variants that create a premature translation termination codon (nonsense codon) account for the most common forms of beta thalassemia, in terms of numbers of patients affected. These variants create translation stop signals prematurely, so that the complete beta globin polypeptide is never made (figure 3). Rather, a truncated fragment of globin is produced. In the most common forms of thalassemia due to this mechanism, these globin fragments are highly unstable, resulting in the accumulation of no detectable protein from the mutated gene locus (beta0 thalassemia). As a result, patients homozygous for this variant cannot make any beta chains and have a severe form of beta thalassemia.
Premature translation termination can occur by base substitution or from short insertions or deletions of DNA in an exon, producing so called "frame shift" mutations. Unless the inserted or deleted stretch of DNA contains a number of bases that is an exact multiple of three, so that the "open" translation reading frame is maintained, the insertion or deletion will cause the ribosome to begin reading the codons out of register (out of the normal reading frame). One consequence of this frame shift mechanism is that the probability that a UAA, UAG, or UGA translation termination codon will be encountered in register with the shifted reading frame within 50 or so bases downstream is almost 100 percent.
In the normal reading frame, these three bases (UAA, UAG, UGA) are usually divided among two codons and thus never read as a stop codon by the ribosome. In the shifted reading frame, the three bases will appear as a single codon and cause the ribosome to cease translation. Frame shifting is thus an alternative molecular pathway to premature translation termination. It accounts for a number of both alpha and beta thalassemia variants.
Nonsense-mediated decay — The physiologic consequences of translation termination are very clear; no functioning globin is produced and thalassemia results. Less obvious is the phenomenon that these prematurely terminated mRNAs accumulate in greatly reduced amounts. It is not obvious why accumulation should be impaired, since their transcription, processing, and stability elements are intact. A great deal of work has shown that there are normal cell mechanisms to eliminate abnormally translated mRNAs. They probably exist to guard against the accumulation of truncated protein products, which have the potential to interact abnormally with other proteins and damage cells. This protective phenomenon is called nonsense-mediated decay and was first described for the globin genes.
Curiously, premature stop codons that occur in the final exon of either the alpha or beta globin gene accumulate at nearly normal levels. Moreover, the truncated polypeptides also accumulate stably and in significant amounts. It appears that the process of nonsense-mediated decay affects only those mRNAs in which the premature stop codon occurs in the first or second exons. This has been found in other gene systems as well. Indeed, there is increasing evidence that nonsense-mediated decay and mRNA splicing are interactive processes [36].
Dominant thalassemia trait due to nonsense codons in the final exon — The consequences of the failure of nonsense-mediated decay to reduce the accumulation of mRNAs carrying nonsense codons in the final exon are illustrated by the instructive phenotype associated with these variants. (See 'Nonsense-mediated decay' above.)
In a few kindreds with either alpha or beta thalassemia, individuals with clinical manifestations of thalassemia are in fact "simple" heterozygotes inheriting only one allele for beta or alpha thalassemia. In another words, they inherit a dominant form of thalassemia.
In every case studied, this dominance with increased clinical severity of the heterozygous state appears to be due to the fact that premature translation termination in the third exon results in the production of a truncated globin polypeptide that is sufficiently stable to form hemoglobin tetramers that are highly unstable. The tetramers precipitate, forming inclusion bodies, and all of the downstream derangements associated with thalassemia occur [37,38]. The globin peptides that prematurely terminate in the third exon thus behave as dominant negative mutations.
FAILED TRANSLATION TERMINATION: Hb CONSTANT SPRING — Hb Constant Spring is a prototype of several pathogenic variants, some relatively common, in which altered translation and mRNA stability interact to produce severe thalassemia. Hb Constant Spring results from a single base substitution that converts the normal UAA translation termination codon in the alpha globin mRNA into a "sense" codon coding for an amino acid. As a result, the ribosome reads through the normal termination site and adds another 31 amino acids before it encounters an in frame stop codon in the normally 3' untranslated region.
The elongated alpha globin polypeptide (alphaCS) that results from this abnormal translation event is functional and relatively stable, although it becomes modestly foreshortened by proteolysis during the life span of the red cell. However, very little alphaCS globin is produced, usually only 1 to 5 percent of the normal output from the alpha globin gene. This thalassemic behavior occurs because the amount of alphaCS mRNA is dramatically reduced, due to its marked instability [39].
Sequence elements in the 3' untranslated region (3' UTR) of normal alpha globin mRNA are critical for its extraordinary stability. These elements are situated within the region that is "read through" in alphaCS mRNA by the ribosome as a result of the loss of the termination codon. This read through phenomenon disrupts the ability of stability elements to interact with their cognate binding proteins, resulting in the destabilization of the alphaCS globin mRNA (figure 4).
Hb Constant Spring is a very common form of the alpha thalassemia in Asian regions. Several analogous variants have been described. Each represents a different point mutation in the termination codon that results in a different amino acid being incorporated where translation should normally stop. In each of these cases, read-through proceeds to the exact same stop signal in the 3' UTR, so that mRNA stability is affected exactly as it is for Hb Constant Spring. Other rare mutations disrupt translation termination by frame shifting. In these cases, the severity of the alteration in mRNA stability varies, probably depending on the point at which a new in frame termination codon is encountered and the way in which the variable lengths of ribosomal read through interferes with the function of the 3' UTR stability elements.
POST TRANSLATIONAL MECHANISMS — A few relatively uncommon variants have been found that cause thalassemia by disrupting the structure of the fully translated globin product. These appear to interfere with normal folding of the globin peptide to form stable dimers or tetramers. In each case, the abnormal globin generates inclusion bodies and produces a thalassemia phenotype. Examples are Hb Indianapolis, which produces beta thalassemia intermedia, and Hb Quong Sze, which produces alpha thalassemia [40]. The final common pathway for these variants is similar to that of the dominant form of thalassemia due to nonsense codons in the final exon. (See 'Dominant thalassemia trait due to nonsense codons in the final exon' above.)
In both cases, substantial amounts of the abnormal protein accumulate, generating inclusion bodies by virtue of their abnormal interaction with other globin chains or heme.
SUMMARY
●Globin genes – Genes coding for the individual globins are located in two clusters (figure 1). The globin genes were the first human genes to be cloned and thoroughly characterized and provided the paradigm for the identification of other single gene disorders. (See 'Globin gene anatomy and physiology' above.)
●Pathogenic variants in thalassemia – Thalassemias arise from well over 100 pathogenic variants that, in the aggregate, affect every step required for successful production of the large amounts of Hb needed for normal red cell homeostasis. Common and uncommon pathogenic variants have been highly informative, because their study has uncovered mechanisms that are highly relevant to the pathophysiology of a number of heritable and acquired disorders. Studies of thalassemia continue to serve as a paradigm for the study of molecular medicine in general. (See 'Molecular lesions causing thalassemia' above and 'Introduction' above.)
•The variants accounting for most of the thalassemia patients around the world are those affecting translation termination and mRNA splicing. (See 'Variants affecting pre-mRNA splicing' above and 'Altered mRNA translation and stability' above and 'Failed translation termination: Hb constant spring' above.)
•Gene deletions and rearrangements, and defects affecting transcription, mRNA stability, or Hb assembly are considerably less common. (See 'Gene deletions' above and 'Gene rearrangements' above.)
ACKNOWLEDGMENT — UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.
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