Protein Analysis

In 1956, by fractionating the peptide products of digestion of human Hb with the proteolytic enzyme, trypsin, Ingram found 30 discrete peptide fragments. Trypsin cuts polypeptide chains at the amino acids arginine and lysine. Analysis of the 580 amino acids of human Hb had previously shown there to be a total of 60 arginine and lysine residues, suggesting that Hb was made up of two identical peptide chains with 30 arginine and lysine residues on each chain.

At about the same time, a family was reported in which two hemoglobin variants, Hb S and Hb Hopkins II, were both present in some family members. Several members of the family who possessed both variants had children with normal Hb, offspring who were heterozygous for only one Hb variant, as well as offspring who, like their parents, were doubly heterozygous for the two Hb variants. These observations provided further support for the suggestion that at least two different genes were involved in the production of human Hb.

Shortly thereafter, the amino-terminal amino acid sequence of human Hb was determined and showed valine–leucine and valine–histidine sequences in equimolar proportions, with two moles of each of these sequences per mole of Hb. This was consistent with human Hb being made up of a tetramer consisting of two pairs of different polypeptides referred to as the α- and β-globin chains.

Analysis of the iron content of human Hb revealed that iron constituted 0.35% of its weight, from which it was calculated that human Hb should have a minimum molecular weight of 16,000 Da. In contrast, determination of the molecular weight of human Hb by physical methods gave values of the order of 64,000 Da, consistent with the suggested tetrameric structure, α₂β₂, with each of the globin chains having its own iron-containing group—heme (Figure 10.1).

FIGURE 10.1 Diagrammatic representation of one of the globin chains and associated porphyrin molecule of human hemoglobin.

Subsequent investigators demonstrated that Hb from normal adults also contained a minor fraction, constituting 2% to 3% of the total Hb, with an electrophoretic mobility different from the majority of human Hb. The main component was called Hb A, whereas the minority component was called Hb A₂. Subsequent studies revealed Hb A₂ to be a tetramer of two normal α chains and two other polypeptide chains whose amino-acid sequence resembled most closely the β chain and was designated delta (δ).

Protein Studies

Amino acid sequencing of the various globin polypeptides in the 1960s showed that the α chain was 141 amino acids long compared with the β chain’s 146 amino acids. Their amino acid sequences were similar but by no means identical. The sequence of the δ chain differs from the β chain by 10 amino acids, and analysis of the γ chain showed that it also most closely resembles the β chain, differing by 39 amino acids. In addition, it was found that there were two types of HbF, in which the γ chain contain either the amino acid glycine or alanine at position 136, designated (G)γ and (A)γ, respectively. Partial sequence analyses of the ζ and ε chains of embryonic Hb suggest that ζ is similar in amino acid sequence to the α chain, whereas ε resembles the β chain.

Thus, there are two groups of globin chains, the α-like and β-like, possibly derived from an ancestral Hb gene that has undergone a number of gene duplications and diverged over evolutionary time.

Globin Gene Mapping

The first evidence for the arrangement of the various globin structural genes on the human chromosomes was provided by analysis of the Hb electrophoretic variant, Hb Lepore. Comparison of trypsin digests of Hb Lepore with Hb from normal people revealed that the α chains were normal, whereas the non-α chains appeared to consist of an amino-terminal δ-like sequence and a carboxy-terminal β-like sequence. It was therefore proposed that Hb Lepore could represent a ‘fusion’ globin chain that had arisen as a result of a crossover coincidental with mispairing of the δ- and β-globin genes during meiosis as a result of the sequence similarity of the two genes and the close proximity of the δ- and β-globin genes on the same chromosome (Figure 10.3). If this hypothesis was correct, it was argued that there should also be an ‘anti-Lepore’ Hb (i.e., a β–δ-globin fusion product in which the non-α-globin chains contained β-chain residues at the amino-terminal end and δ-chain residues at the carboxy-terminal end). In the late 1960s, a new Hb electrophoretic variant, Hb Miyada, was identified in Japan, whereby analysis of trypsin digests showed it to contain β-globin sequence at the amino-terminal end and δ-globin sequence at the carboxy-terminal end, as predicted.

FIGURE 10.3 Mechanism of unequal crossing over which generates Hb Lepore and anti-Lepore.

(Adapted from Weatherall DJ, Clegg JB 1981 The thalassaemia syndromes. Blackwell, Oxford.)

Further evidence at the protein level for the physical mapping of the human globin genes was provided by the report of another Hb electrophoretic variant, Hb Kenya. Amino acid sequence analysis suggested it was a γ–β fusion product with a crossover having occurred somewhere between amino acids 81 and 86 in the two globin chains. For this fusion polypeptide to have occurred, it was argued that the γ-globin structural gene must also be in close physical proximity to the β-globin gene.

Little evidence was forthcoming from protein studies about the mapping of the α-globin genes. The presence of normal Hb A in individuals who, from family studies, should have been homozygous for a particular α chain variant, or obligate compound (double) heterozygotes (p. 114), suggested there could be more than one α-globin gene. In addition, the proportion of the total Hb made up by the α chain variant, in those heterozygous for those variants, was consistently lower (less than 20%) than that seen with the β chain variants (usually more than 30%), suggesting there could be more than one α-globin structural gene.

Globin Gene Structure

The detailed structure of globin genes has been made possible by DNA analysis. Immature red blood cells, reticulocytes, provide a rich source of globin messenger RNA (mRNA) for the synthesis of complementary DNA (cDNA)—reticulocytes synthesize little else! Use of β-globin cDNA for restriction mapping studies of DNA from normal individuals revealed that the non-α, or β-like, globin genes are located in a 50-kilobase (kb) stretch on the short arm of chromosome 11 (Figure 10.4). The entire sequence of this 50-kb stretch containing the various globin structural genes is known. Of interest are non-functional regions with sequences similar to those of the globin structural genes—i.e., they produce no identifiable message or protein product and are pseudogenes.

FIGURE 10.4 The α- and β-globin regions on chromosomes 16 and 11 showing the structural genes and pseudogenes (ψ) and the various hemoglobins produced.

(Adapted from Carrell RW, Lehman H 1985 The haemoglobinopathies. In: Dawson AM, Besser G, Compston N eds. Recent advances in medicine 19, pp. 223–225. Churchill Livingstone, Edinburgh, UK.)

Studies of the α-globin structural genes have shown that there are two α-globin structural genes—α₁ and α₂—located on chromosome 16p (see Figure 10.4). DNA sequencing has revealed nucleotide differences between these two structural genes even though the transcribed α-globin chains have an identical amino acid sequence—evidence for ‘degeneracy’ of the genetic code. In addition, there are pseudo-α, pseudo-ζ, and ζ genes to the 5′ side of the α-globin genes, as well as an additional theta (θ)-globin gene to the 3′ side of the α₁-globin gene. The θ-globin gene, whose function is unknown, is interesting because, unlike the globin pseudogenes, which are not expressed, its structure is compatible with expression. It has been suggested that it could be expressed in very early erythroid tissue such as the fetal liver and yolk sac.

Structural Variants/Disorders

In 1975, Ingram demonstrated that the difference between Hb A and Hb S lay in the substitution of valine for glutamic acid in the β chain. Since then, more than 300 Hb electrophoretic variants have been described due to a variety of types of mutation (Table 10.2). Some 200 of these electrophoretic variants are single amino acid substitutions resulting from a point mutation. The majority are rare and not associated with clinical disease. A few are associated with disease and relatively prevalent in certain populations.

Table 10.2 Structural Variants of Hemoglobin

* Residues are either added (+) or lost (–).

Clinical Aspects

Some Hb variants are associated with disease, but many are harmless and do not interfere with normal function, having been identified coincidentally in the course of population surveys. The more common that interfere with normal Hb function are shown in Table 10.3.

Table 10.3 Functional Abnormalities of Structural Variants of Hemoglobin

Clinical Features	Examples
Hemolytic Anemia
Sickling disorders	HbS/S, HbS/C disease, or HbS/O (Arab), HbS/D (Punjab), HBS/β-thalassemia, HbS/Lepore
	Other rare homozygous sickling mutations—HbS-Antilles, Hb S-Oman)
Unstable hemoglobin	Hb Köln
	Hb Gun Hill
	Hb Bristol
Cyanosis
Hemoglobin M (methemoglobinemia)	Hb M (Boston)
	Hb M (Hyde Park)
Low oxygen affinity	Hb Kansas
Polycythemia
High oxygen affinity	Hb Chesapeake
	Hb Heathrow

If the mutation is on the inside of the globin subunits, in close proximity to the heme pockets, or at the interchain contact areas, this can produce an unstable Hb molecule that precipitates in the red blood cell, damaging the membrane and resulting in hemolysis of the cell. Alternatively, mutations can interfere with the normal oxygen transport function of Hb, leading to either enhanced, or reduced, oxygen affinity, or an Hb that is stable in its reduced form, so-called methemoglobin.

The structural variants of Hb identified by electrophoretic techniques probably represent a minority of the total number of variants that exist, as it is predicted that only one-third of the possible Hb mutations that could occur will produce an altered charge in the Hb molecule, and thereby be detectable by electrophoresis (Figure 10.5).

FIGURE 10.5 Hemoglobin electrophoresis showing hemoglobins A, C, and S.

(Courtesy Dr. D. Norfolk, General Infirmary, Leeds, UK.)

Sickle Cell Disease

This severe hereditary hemolytic anemia was first recognized clinically early in the twentieth century, but in 1940 red blood cells from affected individuals with sickle cell (SC) disease were noted to appear birefringent when viewed in polarized light under the microscope, reflecting polymerization of the sickle hemoglobin. This distorts the shape of red blood corpuscles under deoxygenated conditions—so-called sickling (Figure 10.6). Pauling, in 1949, using electrophoresis, showed that it had different mobility to HbA and called it HbS, for sickle.

FIGURE 10.6 Blood film showing sickling of red cells in sickle-cell disease. Sickled cells are arrowed.

(Courtesy Dr. D. Norfolk, General Infirmary, Leeds, UK.)

Clinical Aspects of SC Disease

SC disease, following autosomal recessive inheritance, is the most common hemoglobinopathy; in the United Kingdom, about 310,000 individuals are carriers, and approximately 400 pregnancies are affected annually. The disease is especially prevalent in those areas of the world where malaria is endemic. The parasite Plasmodium falciparum is disadvantaged because the red cells of SC heterozygotes are believed to express malarial or altered self-antigens more effectively, resulting in more rapid removal of parasitized cells from the circulation. SC heterozygotes are therefore relatively protected from malarial attacks. They are therefore biologically fitter, the SC gene can be passed on to the next generation, and over time this has resulted in relatively high gene frequency in malarial-infested regions (see Chapter 8). The clinical manifestations are manifold and include painful sickle cell crisis, chest crisis, aplastic crisis, splenic sequestration crisis, priapism, retinal disease, and cerebrovascular accident. Pulmonary hypertension may occur and heart failure can accompany severe anemia during aplastic or splenic sequestration crises. All of these are the result of deformed, sickle-shaped red cells, which are less able to change shape and tend to obstruct small arteries, thus reducing oxygen supply to the tissues (Figure 10.7). Sickled cells, with damaged cell membranes, are taken up by the reticuloendothelial system. The shorter red cell survival time leads to a more rapid red cell turnover and, consequently, anemia.

FIGURE 10.7 The pleiotropic effects of the gene for sickle-cell disease.

Sickling crises reduce life expectancy, so early recognition and treatment of the complications is vital. Prophylactic penicillin to prevent the risk of overwhelming sepsis from splenic infarction has been successful and increased survival. The other beneficial approach is the use of hydroxyurea, a simple chemical compound that can be taken orally. Once-daily administration has been shown to increase levels of HbF through pharmacological induction. The HbF percentage has been shown to predict the clinical severity of SC disease, preventing intracellular sickling, which decreases vasoocclusion and hemolysis. It has been suggested that a potential threshold of 20% HbF is required to prevent recurrent vasoocclusive events. Hydroxyurea is well-tolerated, safe, and has many features of an ideal drug. The US Food and Drug Administration approved hydroxyurea for adult patients with clinically severe some years ago, but it has been used only sparingly. There is ongoing debate about its wider use in less severe cases and children.

Disorders of Hemoglobin Synthesis

The thalassemias are the commonest single group of inherited disorders in humans, occurring in persons from the Mediterranean region, Middle East, Indian subcontinent, and Southeast Asia. They are heterogeneous and classified according to the particular globin chain, or chains, synthesized in reduced amounts (e.g., α-, α-, δβ-thalassemia).

There are similarities in the pathophysiology of all forms of thalassemia, though excessive α chains are more haemolytic than excessive β chains. An imbalance of globin-chain production results in the accumulation of free globin chains in the red blood cell precursors which, being insoluble, precipitate, resulting in hemolysis of the red blood cells (i.e., a hemolytic anemia). The consequence is compensatory hyperplasia of the bone marrow.

α-Thalassemia

This results from underproduction of the α-globin chains and occurs most commonly in Southeast Asia but is also prevalent in the Mediterranean, Middle East, India, and sub-Saharan Africa, with carrier frequencies ranging from 15% to 30%. There are two main types of α-thalassemia, with different severity: the severe form, in which no α chains are produced, is associated with fetal death due to massive edema secondary to heart failure from severe anemia—hydrops fetalis (Figure 10.8). Analysis of Hb from such fetuses reveals a tetramer of γ chains, originally called Hb Barts.

FIGURE 10.8 Longitudinal ultrasonographic scan of a coronal section of the head (to the right) and thorax of a fetus with hydrops fetalis from the severe form of α-thalassemia, Hb Barts, showing a large pleural effusion (arrows).

(Courtesy Mr. J. Campbell, St. James’s Hospital, Leeds, UK.)

In the milder forms of α-thalassemia compatible with survival, although some α chains are produced, there is still a relative excess of β chains, resulting in production of the β-globin tetramer Hb H—known as Hb H disease. Both Hb Barts and Hb H globin tetramers have an oxygen affinity similar to that of myoglobin and do not release oxygen as normal to peripheral tissues. Also, Hb H is unstable and precipitates, resulting in hemolysis of red blood cells.

Mutational basis of α-thalassemia

The absence of α chain synthesis in hydropic fetuses, and partial absence in Hb H disease, was confirmed using quantitative mRNA studies from reticulocytes. Studies comparing the quantitative hybridization of radioactively labeled α-globin cDNA to DNA from hydropic fetuses, and in Hb H disease, were consistent with the α-globin genes being deleted. Restriction mapping studies of the α-globin region revealed two α-globin structural genes on chromosome 16p. The various forms of α-thalassemia are to be mostly the result of deletions of one or more of these structural genes (Figure 10.9). These deletions are thought to have arisen as a result of unequal crossover events in meiosis, more likely to occur where genes with homologous sequences are in close proximity. Support for this hypothesis comes from the finding of the other product of such an event (i.e., individuals with three α-globin structural genes located on one chromosome).

FIGURE 10.9 Structure of the normal and deleted α-globin structural genes in the various forms of α-thalassemia.

(Adapted from Emery AEH 1984 An introduction to recombinant DNA. John Wiley, Chichester.)

These observations resulted in the recognition of two other milder forms of α-thalassemia that are not associated with anemia and can be detected only by the transient presence of Hb Barts in newborns. Mapping studies of the α-globin region showed that these milder forms of α-thalassemia are due to the deletion of one or two of the α-globin genes. Occasionally, non-deletion point mutations in the α-globin genes, as well as the 5′ transcriptional region, have been found to cause α-thalassemia.

An exception to this classification of α-thalassemias is the Hb variant Constant Spring, named after the town in the United States from which the original patient came. This was detected as an electrophoretic variant in a person with Hb H disease. Hb Constant Spring is due to an abnormally long α chain resulting from a mutation in the normal termination codon at position 142 in the α-globin gene. Translation of α-globin mRNA continues until another termination codon is reached, resulting in an abnormally long α-globin chain. The abnormal α-globin mRNA molecule is also unstable, leading to a relative deficiency of α chains and the presence of the β-globin tetramer, Hb H.

β-Thalassemia

By now the reader will deduce that this is caused by underproduction of the β-globin chain of Hb. Production of β-globin chains may be either reduced (β⁺) or absent (β⁰). Individuals homozygous for β⁰-thalassemia mutations have severe, transfusion-dependent anemia. Approximately 1:1000 Northern Europeans are β-thalassemia carriers and in United Kingdom, on average, 22 babies with β⁰ thalassemia are born annually and roughly 860 people live with the condition; there are an estimated 327,000 carriers.

Mutational basis of β-Thalassemia

β-Thalassemia is rarely the result of gene deletion and DNA sequencing is often necessary to determine the molecular pathology. In excess of 100 different mutations have been shown to cause β-thalassemia, including point mutations, insertions, and base-pair deletions. These occur within both the coding and non-coding portions of the β-globin genes as well as the 5′ flanking promoter region, the 5′ capping sequences (p. 19) and the 3′ polyadenylation sequences (p. 19) (Figure 10.10). The various mutations are often unique to certain population groups and can be considered to fall into six main functional types.

FIGURE 10.10 Location and some of the types of mutation in the β-globin gene and flanking region that result in β-thalassemia.

(Adapted from Orkin SH, Kazazian HH 1984 The mutation and polymorphism of the human β-globin gene and its surrounding DNA. Annu Rev Genet 18:131–171.)

Transcription mutations. Mutations in the 5′ flanking TATA box or the promoter region of the β-globin gene can result in reduced transcription levels of the β-globin mRNA.

mRNA splicing mutations. Mutations involving the invariant 5′ GT or 3′ AG dinucleotides of the introns in the β-globin gene or the consensus donor or acceptor sequences (p. 19) result in abnormal splicing with consequent reduced levels of β-globin mRNA. The most common Mediterranean β-thalassemia mutation leads to the creation of a new acceptor AG dinucleotide splice site sequence in the first intron of the β-globin gene, creating a ‘cryptic’ splice site (p. 25). The cryptic splice site competes with the normal splice site, leading to reduced levels of the normal β-globin mRNA. Mutations in the coding regions of the β-globin region can also lead to cryptic splice sites.

Polyadenylation signal mutations. Mutations in the 3′ end of the untranslated region of the β-globin gene can lead to loss of the signal for cleavage and polyadenylation of the β-globin gene transcript.

RNA modification mutations. Mutations in the 5′ and 3′ DNA sequences, involved respectively in the capping (p. 19) and polyadenylation (p. 19) of the mRNA, can result in abnormal processing and transportation of the β-globin mRNA to the cytoplasm, and therefore reduced levels of translation.

Chain termination mutations. Insertions, deletions, and point mutations can all generate a nonsense or chain termination codon, leading to premature termination of translation of the β-globin mRNA. Usually this results in a shortened β-globin mRNA that is unstable and more rapidly degraded, again leading to reduced levels of translation of an abnormal β-globin.

Missense mutations. Rarely, missense mutations lead to a highly unstable β-globin (e.g., Hb Indianapolis).

Clinical aspects of β-thalassemia

Children with thalassemia major, or Cooley’s anemia as it was originally known, usually present in infancy with a severe transfusion-dependent anemia. Unless adequately transfused, compensatory expansion of the bone marrow results in an unusually shaped face and skull (Figure 10.11). Affected individuals used to die in their teens or early adulthood from complications resulting from iron overload from repeated transfusions. However, daily use of iron-chelating drugs, such as desferrioxamine, has greatly improved their long-term survival.

FIGURE 10.11 Facies of a child with β-thalassemia showing prominence of the forehead through changes in skull shape as a result of bone marrow hypertrophy.

(Courtesy Dr. D. Norfolk, General Infirmary, Leeds, UK.)

Individuals heterozygous for β-thalassemia—thalassemia trait or thalassemia minor—usually have no symptoms or signs but do have a mild hypochromic, microcytic anemia. This can easily be confused with iron deficiency anemia.

δβ-Thalassemia

In this hemoglobinopathy, there is underproduction of both the δ and β chains. Homozygous individuals produce no δ- or β-globin chains, which one might expect to cause a profound illness. However, they have only mildly anemia because of increased production of γ chains, with Hb F levels being much higher than the mild compensatory increase seen in β⁰ thalassemia.

Mutational basis of δβ-thalassemia

The cause is extensive deletions in the β-globin region involving the δ- and β-globin structural genes (Figure 10.12). Some large deletions include the Aγ-globin gene so that only the Gγ-globin chain is synthesized.

FIGURE 10.12 Some of the deletions in the β-globin region that result in some forms of thalassemia and hereditary persistence of fetal hemoglobin.