Thalassemia is a significant health problem worldwide. Prenatal diagnosis is the only effective way to prevent the birth of a fetus with severe thalassemias, which include hemoglobin Bart’s hydrops fetalis and thalassemia major. However, accurate prenatal diagnosis depends on the comprehensive consideration of the molecular basis of thalassemias. To make a correct decision, the obstetrician should have a certain understanding of the genetics of thalassemias. Here we present a brief introduction of some fundamental genetic knowledge of thalassemias, including the production of hemoglobin, structure and location of globin genes, hemoglobin switch, epidemiology, clinical classification, molecular and cellular pathology, genotype–phenotype correlation, and genetic modifiers. Furthermore, some unusual clinical cases that cannot be explained by Mendel’s laws are described. On the basis of a thorough understanding of the above information, clinicians should have the ability to precisely diagnose thalassemia patients and provide applicable genetic counselling to the affected families.
Highlights
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Thalassemia is a significant health problem worldwide.
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Hb Bart’s hydrops fetalis and TM should be prevented.
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Identification of genetic modifiers provides new information for the diagnosis.
Introduction
Inherited hemoglobin (Hb) disorders are the most common inherited blood disorders globally and account for approximately 3.4% of deaths in children under 5 years of age . This group of diseases is caused by mutations in human globin genes, which are classified into two categories: those that produce structurally abnormal globin (Hb variants) and those with impaired globin synthesis (thalassemia). Thalassemia is characterized by the absence or decreased accumulation of one of the globin subunits. The most common forms are α-thalassemia (OMIM: # 604131 ) and β-thalassemia (OMIM: # 613985 ), which affect the synthesis of α- and β-globin subunits, respectively.
Thalassemias are prevalent in tropical and subtropical areas where malaria was and still is epidemic. The high frequency may be due to carriers of hemoglobinopathies who have a survival advantage in malarial endemic areas . People carrying thalassemia variants are concentrated in Southeast Asia, the Mediterranean area, the Indian subcontinent, the Middle East, and Africa . Moreover, it is noteworthy that as a consequence of recent massive population migrations, thalassemia is not restricted to traditional high-incidence regions and is now a relatively common clinical problem in North America, North Europe, and Australia . The clinical management of thalassemia, such as its diagnosis and treatment, has challenged the local health system. For example, screening for Hb H disease (one form of α-thalassemia) has been added for newborns in California . An analysis of 86 Hb H disease patients performed by Lal’s group supported the usefulness of universal newborn screening and suggested that the screening should be extended to other populations .
The inheritance mode of thalassemias is autosomal recessive (AR). Carriers of thalassemia mutations are clinically normal. However, when both parents are carriers, for every pregnancy, there is a 25% chance that the child will be a thalassemia patient, a 50% chance that the child will be a thalassemia carrier, and a 25% chance that the child will be normal. To date, prenatal diagnosis is the only way to prevent the birth of an affected child. Therefore, in highly prevalent regions, an ideal and effective strategy to decrease the birth rate of thalassemia patients is to identify high-risk couples, who are both carriers, before pregnancy by screening (or carrier testing) and then perform a prenatal diagnosis during pregnancy.
Basic genetic structures of Hemoglobin gene clusters
All normal human Hbs are tetramers of two pairs of globin chains: one pair of α-like globins and one pair of β-like globins. At the molecular level, Hb synthesis is controlled by two multigene clusters ( Fig. 1 A). The α-cluster contains an embryonic gene (ζ2), two fetal/adult α genes (α2 and α1), two pseudo genes (Ψζ1 and Ψα1), and two minor globin-like genes (Ψα2 and θ), which are all arranged in the following order: 5′-ζ2–Ψζ1–Ψα2–Ψα1–α2–α1–θ-3′. HS-40 is the major regulatory element of the α-globin locus. The β-cluster contains an embryonic gene (ɛ), two fetal genes ( G γ and A γ), one pseudo gene (Ψβ), and two adult genes (δ and β), which are arranged in the following order: 5′-ɛ– G γ– A γ–Ψβ–δ–β–3′. Locus control region (LCR) is the important upstream regulatory region.
Clinically, thalassemias display a wide spectrum of phenotypes ranging from asymptomatic to lethal. According to the clinical severity, thalassemias are generally divided into three groups: (1) Thalassemia trait: they are carriers who are often asymptomatic and do not need any treatment. (2) Thalassemia intermedia (TI): they have moderate anemia (Hb 60–100 g/L) and occasionally require red blood cell transfusion; in α-thalassemia, it is known as Hb H disease. (3) Thalassemia major (TM): they have severe anemia and require transfusions for survival; in α-thalassemia, this clinical form was named Hb Bart’s hydrops fetalis. The fetus usually dies in utero or shortly after birth. In general, the latter two groups are defined as thalassemia patients. The common symptoms of these patients include pallor, jaundice, splenomegaly, and skeletal deformities.
The imbalance of subunits is central to the pathophysiology of thalassemia. For normal Hb synthesis, the ratio of α:non-α subunits should be 1:1 ( Fig. 1 B). In α-thalassemia, the quantity of β-like chains is greater than that of α chains; on the contrary, in β-thalassemia, the quantity of β-like chains is less than that of α chains. The degree of imbalance is proportional to the severity of the disease. In Hb Bart’s hydrops fetalis, because of the absence of the α-globin, fetal blood contains mainly Hb Bart (γ4). Hb Bart’s cannot release oxygen even in a hypoxic state. Thus, the fetus suffers from severe anemia and hypoxia and often develops fetal anomalies. Such a fetus always dies either in utero (23–38 weeks) or shortly after birth. This disease causes up to 90% of all fetal hydrops in Southeast Asia . In Hb H disease, which is the intermedia α-thalassemia clinical form, the affected individual usually produces less than 30% of the normal amount of α-globin, whereas a relative excess of β-globin chains form Hb H (β4). Hb H is unstable and precipitates inside the red cells, which are prematurely destroyed, resulting in moderate hemolysis. The main pathophysiological mechanisms of β-thalassemia are hemolysis and ineffective erythropoiesis. Insufficiency of β-globin results in an excess of free α-globin. The unstable free α-globin forms α-hemichromes, generates reactive oxygen species (ROS), and triggers cascades of events that lead to hemolysis and ineffective erythropoiesis. Further complications include iron overload, splenomegaly, skeletal deformities, and erythroid marrow expansion.
Basic genetic structures of Hemoglobin gene clusters
All normal human Hbs are tetramers of two pairs of globin chains: one pair of α-like globins and one pair of β-like globins. At the molecular level, Hb synthesis is controlled by two multigene clusters ( Fig. 1 A). The α-cluster contains an embryonic gene (ζ2), two fetal/adult α genes (α2 and α1), two pseudo genes (Ψζ1 and Ψα1), and two minor globin-like genes (Ψα2 and θ), which are all arranged in the following order: 5′-ζ2–Ψζ1–Ψα2–Ψα1–α2–α1–θ-3′. HS-40 is the major regulatory element of the α-globin locus. The β-cluster contains an embryonic gene (ɛ), two fetal genes ( G γ and A γ), one pseudo gene (Ψβ), and two adult genes (δ and β), which are arranged in the following order: 5′-ɛ– G γ– A γ–Ψβ–δ–β–3′. Locus control region (LCR) is the important upstream regulatory region.
Clinically, thalassemias display a wide spectrum of phenotypes ranging from asymptomatic to lethal. According to the clinical severity, thalassemias are generally divided into three groups: (1) Thalassemia trait: they are carriers who are often asymptomatic and do not need any treatment. (2) Thalassemia intermedia (TI): they have moderate anemia (Hb 60–100 g/L) and occasionally require red blood cell transfusion; in α-thalassemia, it is known as Hb H disease. (3) Thalassemia major (TM): they have severe anemia and require transfusions for survival; in α-thalassemia, this clinical form was named Hb Bart’s hydrops fetalis. The fetus usually dies in utero or shortly after birth. In general, the latter two groups are defined as thalassemia patients. The common symptoms of these patients include pallor, jaundice, splenomegaly, and skeletal deformities.
The imbalance of subunits is central to the pathophysiology of thalassemia. For normal Hb synthesis, the ratio of α:non-α subunits should be 1:1 ( Fig. 1 B). In α-thalassemia, the quantity of β-like chains is greater than that of α chains; on the contrary, in β-thalassemia, the quantity of β-like chains is less than that of α chains. The degree of imbalance is proportional to the severity of the disease. In Hb Bart’s hydrops fetalis, because of the absence of the α-globin, fetal blood contains mainly Hb Bart (γ4). Hb Bart’s cannot release oxygen even in a hypoxic state. Thus, the fetus suffers from severe anemia and hypoxia and often develops fetal anomalies. Such a fetus always dies either in utero (23–38 weeks) or shortly after birth. This disease causes up to 90% of all fetal hydrops in Southeast Asia . In Hb H disease, which is the intermedia α-thalassemia clinical form, the affected individual usually produces less than 30% of the normal amount of α-globin, whereas a relative excess of β-globin chains form Hb H (β4). Hb H is unstable and precipitates inside the red cells, which are prematurely destroyed, resulting in moderate hemolysis. The main pathophysiological mechanisms of β-thalassemia are hemolysis and ineffective erythropoiesis. Insufficiency of β-globin results in an excess of free α-globin. The unstable free α-globin forms α-hemichromes, generates reactive oxygen species (ROS), and triggers cascades of events that lead to hemolysis and ineffective erythropoiesis. Further complications include iron overload, splenomegaly, skeletal deformities, and erythroid marrow expansion.
Hemoglobin switch process
Because all normal Hbs are tetramers of two pairs of globin chains, the production of α-like and β-like chains is balanced in each stage of development. The structure of human Hb changes during development, as shown in Fig. 2 . In the embryonic stage, there are Hb Gower1 (ζ2ɛ2), Hb Gower2 (α2ɛ2), and Hb Portland (ζ2γ2). These embryonic Hbs are confined to the yolk-sac stage and thereafter are replaced by the fetal hemoglobin (Hb F) (α2γ2). Hb F is the dominant Hb in utero. After birth, Hb F is replaced by adult hemoglobin (Hb A) (α2β2, major Hb, approximately 97%) and Hb A 2 (α2δ2, minor Hb approximately 2%–3%) over the first year of life. Hb F is present during the first 6 months such that the babies do not develop β-thalassemia at birth. In normal adults, Hb F continues to be present, constituting approximately 1% of the total Hb . The entire process is called Hb switch. It is very interesting that the globin genes in the two clusters are arranged along the chromosome in the same order ( Fig. 1 ) according to which they are expressed during development. The sequential activation and silencing of globin genes are precisely controlled. Studies of the expression of globin genes had shown that HS-40 in α-cluster and LCR in β-cluster play important roles as cis-regulating elements . Each element is bound by the complex formed by many proteins that serve as trans-acting factors.
The gene switch in the α-cluster is relatively simple. The two α genes are continuously expressed throughout life, except for the first 6 weeks of embryogenesis in which ζ protein is produced. In contrast, the gene switch in the β-cluster is more complex. It consists of a change from ɛ→γ→β. In particular, the γ to β transition is more important in clinical practice because a high level of Hb F could be curative for β-thalassemia. How to reactivate the γ-genes to bind the excess α-globin is one of the main directions of thalassemia treatment. Studies have established two major mechanisms for γ silencing in adults, including the competitive interaction of the γ- and β-genes with the LCR during the fetal to adult switch and gene-autonomous γ-globin silencing . The latter mechanism provides the basis for a gene-based strategy to increase the Hb F level after birth to cure patients with TM. Many transcription factors are involved in this mechanism, including BCL11A, KLF1, MYB, LRF, and others ( Fig. 3 ).
BCL11A acts as a major repressor of γ-globin expression. Loss of function of BCL11A, regardless of whether it occurs in human erythroid precursor or in transgenic mice, is sufficient to prevent γ-globin repression . It appears to exert its repressive function at a distance. BCL11A binds to the LCR, but it does not bind to the γ-globin or β-globin gene themselves . BCL11A is necessary for configuring the β-locus. It promotes long-range interactions between the LCR and β-globin gene. Thus, when BCL11A is knocked out, the LCR interacts with γ-globin genes instead of the β-globin gene, and the γ-globin expression is reactivated.
KLF1 is a master regulator of adult β-globin transcription. Inactivation of the Klf1 gene in mice showed that it is essential for the activation of β-globin expression . It also mediates the γ to β switch by binding the BCL11A gene promoter and activating its transcription. Knocking down the expression of KLF1 inhibited the expression of BCL11A gene and increased the γ:β ratio in erythroblasts . It is suggested that the KLF1/BCL11A regulatory axis plays a crucial role in the Hb switch . In the normal development process, KLF1 activates BCL11A, which next represses γ gene expression, thereby promoting the switch from Hb F (α2γ2) to Hb A (α2β2). At the same time, KLF1 itself activates β-globin expression. In some cases of hereditary persistence of fetal hemoglobin (HPFH), a haplo-insufficiency of KLF1 results in reduced BCL11A expression, which in turn increases the Hb F level and decreases the Hb A level.
The mechanism of MYB in affecting γ-globin expression is still not clear. However, the disruption of MYB in mice produced an increase in ɛ- and γ-globin expression, indicating that MYB accounts for γ-globin silencing during development . Recently, LRF has been identified as a new transcription factor that represses γ-globin expression . It interacts with the γ-globin genes and maintains the nucleosome density necessary for γ-globin gene silencing in adults. The function of LRF repressing γ-globin is not dependent on BCL11A protein; this suggests that there may be more elements or factors contributing to the Hb switch. Epigenetics alteration and microRNAs should be considered in the future.
Molecular basis of pathogenesis of thalassemia
Thalassemias are caused by two types of defects in globin genes: deletion defects and nondeletion defects. The range of deletion defects usually involves more than 1 kb. Nondeletion defects consist of single nucleotide substitutions or oligonucleotide deletions/insertions. It is interesting that a different spectrum of α- and β-thalassemia mutations is often found in different populations, although thalassemia is a common worldwide disorder. Therefore, reference data of the mutations found in specific populations are characteristics of these populations. When performing molecular diagnosis, the ethnic origin of the patients should be a concern.
α-thalassemia
The vast majority of α-thalassemia is caused by deletion. Because there are two α-globin genes in one chromosome, the haplotype can be written as αα/. Considering one haplotype, α-thalassemia mutations are classified into three groups : (1) α + -thalassemia deletion (-α/), which removes only one α-globin gene; (2) α 0 -thalassemia deletion (–/), which removes both α-globin genes; and (3) nondeletion mutation (α T α/ or αα T /, depending on whether the α2 or α1 gene is affected). The common mutations are listed in Table 1 . The output from the α2 gene accounts for two-thirds of the production of the whole α-globin, whereas the α1 gene accounts for the remaining one-third. Thus, α2 gene mutations would have more severe effects than α1 gene mutations. In addition, the nondeletion may give rise to a more severe reduction in α-chain synthesis than the α + deletion. According to these rules, a haplotype order was established on the basis of its relative effect on α-globin production: αα T /<-α/<α T α/<–/.
| Locus | Mutation/deletion types | Common mutations |
|---|---|---|
| α-globin | α 0 -deletion | — SEA (Southeast Asia), — MED (Mediterranean) |
| α + -deletion | -α 3.7 , -α 4.2 (worldwide) | |
| α T α (α2 gene) | Hb CS (Southeast Asia), α IVS1(−5nt) α (Mediterranean), α PA(AATAAG) α (Middle East Asia) | |
| αα T (α1 gene) | Hb Q-Thailand (Southeast Asia) | |
| β-globin | β ++ -mutation | β −101(C>T) (Mediterranean) |
| β + -mutation | β IVS1–110(G>A) (Mediterranean), Hb E (Southeast Asia) | |
| β 0 -mutation | β CD39(C>T) (Mediterranean), β CD41–42(−CTTT) (Southeast Asia) | |
| deletion (β gene) | 619 bp deletion (Asian Indian) | |
| Deletion (HPFH/ζβ) | SEA-HPFH, Chinese G γ + ( A γδβ) 0 deletion (Chinese) |
More than 40 different α 0 deletions have been reported , the most common being the – SEA (Southeast Asia) and – MED (Mediterranean) mutations. These deletions can be grouped into those that lie entirely within the α-globin cluster (e.g., — SEA ) and deletions that extend to the telomere of chromosome 16 (e.g., — 235 ) . Although — 235 deletions delete other genes, the affected heterozygotes appear phenotypically normal apart from their α-thalassemia . Another type of rare deletions causing α 0 -thalassemia removes the regulatory region HS-40 and leaves the α-globin genes intact . More than 10 α + deletions have been reported; –α 3.7 and –α 4.2 are the most common worldwide . These deletions are the products of reciprocal recombination. These recombinational events also generate α-triplication ααα anti3.7 and ααα anti4.2 . Further recombination events may even result in quadruplicated α genes (αααα) or quintuplicated (ααααα) . Such deletions can also be generated by other molecular mechanisms such as nonhomologous recombination and replication errors . Furthermore, more complex crossover events occur in this cluster, such as “patchwork” α2 and α1 genes (α212 and α121) , the HKαα allele (a rearrangement that contains both the -α 3.7 and ααα anti4.2 unequal crossover junctions), and the anti-HKαα allele (the reciprocal product that contains both the -α 4.2 and ααα anti3.7 unequal crossover junctions) .
At least 90 nondeletion mutations have been found, including mutations that affect mRNA processing, mRNA translation, and α-globin stability. Some Hb variants causing similar thalassemia phenotype were also classified into this group, such as Hb Constant Spring (Hb CS, α CS α) and Hb Quong Sze (Hb QS, α QS α) . Among the currently known nondeletion mutations, the majority of them occur in the α2 gene, and mutations in the α1 gene are rare. The most common nondeletion mutations are α IVS1(−5nt) α (Mediterranean), α PA(AATAAG) α (Middle East Asia), and α Constant Spring α (Southeast Asia) . The Hb Q-Thailand, a G>C mutation in codon 74, is a relatively common α1 mutation in Southeast Asia .
β-thalassemia
In contrast to α-thalassemia, nondeletion defects account for the vast majority of β-thalassemia. More than 300 nondeletion variants have been described in different populations ( http://globin.bx.psu.edu/hbvar/ ). Most of them are point mutations, and only a minority of them are small deletions in the exons of the β-globin gene, such as β CD54–58(−13 bp) and β CD89–93(−14 bp) .
Depending on the degree of quantitative reduction in the synthesis of normal β-globin, β-thalassemia mutations are classified into three groups: (1) β 0 -thalassemia mutation (β 0 /), which results in the absence of β-globin; (2) β + -thalassemia mutation (β + /), which severely reduces the output of β-globin; and (3) β ++ -thalassemia mutation (β ++ /, also known as silent β-mutation), which mildly reduces the output of β-globin. The common β-mutations are also listed in Table 1 . Some Hb variants are synthesized at a reduced rate or are highly unstable and lead to thalassemia phenotypes, such as Hb E (β CD26(G>A) ). This mutation occurs in β-codon 26 (GAG>AAG) and results in amino acid substitution from Glu to Lys; it also activates a new splice site that causes abnormal mRNA processing. It is usually considered a β + -thalassemia mutation . According to the mechanism by which they affect the β-globin gene’s function, the mutations are divided into different groups: (1) mutants that affect transcription, such as β −101(C>T) in the promoter or β CAP+39(C>T) in the 5′UTR ; (2) mutants that affect RNA processing, such as β IVS1–110(G>A) that generate cryptic splice sites, β PA(GATAAG) that decreases the efficiency of the cleavage-polyadenylation process, and β Term CD+32(A>C) in the 3′UTR ; and (3) mutants that affect RNA translation, such as initiation codon mutation β (ATG>GTG) , nonsense mutation β CD39(C>T) , and frameshift mutation β CD41-42(-CTTT) .
Rare deletion mutations of β-thalassemia have also been identified. One group of deletions is restricted to the β-globin gene itself. For example, the 619 bp deletion removes the 3′-end of the β-globin gene. It is a common mutation in Asian Indians and accounts for approximately 30% of the β-thalassemia cases in this population . This group of deletions is also often termed β 0 -mutations. The other group of deletions consists of large deletions involving a part of or the entire β-globin gene clusters. These large deletions are responsible for δβ-thalassemias or HPFH. These large deletions are often associated with an absence of β-globin chain production but are associated with the production of a high quantity of γ-globin. Thus, δβ-thalassemias and HPFH are clinically milder than the typical cases of β 0 -thalassemia . More than 40 large deletions have been reported in different populations. SEA-HPFH and the Chinese G γ + ( A γδβ) 0 deletion are common in southern Chinese populations .
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