Genetic Control of Hemoglobin Synthesis and the Thalassemia Syndromes

Genetic Control of Hemoglobin Synthesis and the Thalassemia Syndromes ARTHUR BANK, M.D. '"

During the past two decades, significant advances have been made in understanding the genetic control of hemoglobin synthesis. I. ":l. "9 The development of new methods of determining hemoglobin structure has been a most significant contribution in this area. IO • 'IC> At present, close to 150 structural abnormalities of hemoglobin have been recognized, most of which differ from normal adult hemoglobin by only a single amino acid. Knowledge of these mutants, together with the recent elucidation of the three-dimensional structure of hemoglobin, has made possible a correlation of the structure and function of this molecule.'l" In addition to information on structurally abnormal hemoglobins, insight has recently been gained into another group of abnormalities of hemoglobin, the thalassemia syndromes.'ll. 41 In these diseases, diminished production of structurally normal hemoglobin occurs; there is a quantitative rather than qualitative change in hemoglobin synthesis. This review will summarize: (1) the structure and genetics of human hemoglobins; (2) control mechanisms in hemoglobin synthesis; and (3) abnormalities in hemoglobin synthesis in the thalassemia syndromes.

STRUCTURE AND GENETICS OF HUMAN HEMOGLOBINS Normal human hemoglobins are composed of two pairs of identical polypeptide or globin chains, each of which consists of between 141 and From the Department of Medicine, Columbia University, College of Physicians and Surgeons, and the Medical Service, Presbyterian Hospital, New York, New York "Assistant Professor of Medicine, Columbia University, College of Physicians and Surgeons, and Leukemia Society Scholar "*Professor of Medicine and Director of Hematology, Columbia University, College of Physicians and Surgeons Supported by grants from the National Institute of Health (GM-14552), National Science Foundation (GB-4631), and Cooley's Anemia Foundation,

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146 amino acids. Each globin chain is linked to a heme group and folded in a manner which permits oxygen transport. Normal Embryonic and Fetal Hemoglobins In early fetal development, two embryonic hemoglobins are present: Hemoglobin Gower I is composed of four 10 chains, and hemoglobin Gower 11 of two 10 and two a chains. 21 In addition, small amounts of another non-a chain containing hemoglobin, hemoglobin Portland, have been found in several normal and abnormal newborns. IH The presence of this hemoglobin, and of hemoglobin Gower I (El) suggests that a chains may be relatively deficient in early fetal life. Subsequently, during gestation, synthesis of fetal hemoglobin (or hemoglobin F) begins and persists until shortly after birth. Hemoglobin F is composed of two a and two y chains, and comprises more than 90 per cent of the total hemoglobin at birth. Its resistance to denaturation by alkali permits its quantitation in the laboratory. Normal Adult Hemoglobins Normal adult hemoglobin or hemoglobin A is composed of two a and two f3 chains and is the major component of adult red blood cells. A relatively minor hemoglobin component in red cells, comprising less than 2 per cent of the total hemoglobin, is hemoglobin A2 , composed of two a and two 0 chains. The f3 chains are closely related in structure to the 0 chains, differing in only 9 of over 140 amino acid residues present in the two molecules. The close structural similarity of these two chains and of other mammalian globin chains has led to the hypothesis of a common ancestral globin gene; the different hemoglobins may result from mutations in this ancestral gene and subsequent duplication of these genes during the process of evolution. 22 Abnormal Human Hemoglobins Although it is beyond the scope of this review to discuss in detail the properties of every mutant hemoglobin, certain principles derived from studies of these abnormal molecules can be summarized. The only differences in hemoglobins so far discovered reside in changes in the globin chain; the heme groups of all human hemoglobins studied to date are identical. Amino acid substitutions in certain positions along the peptide chain result in instability of the hemoglobin molecule. In hemoglobin Zurich, for example, the particular amino acid substitution in the f3 chain (Table 1) results in an alteration of the normal contacts between the heme groups and the globin chains in the hemoglobin molecule.'" The presence of this unstable hemoglobin predisposes the cells containing it to preferential hemolysis when the cells are exposed to certain drugs in vivo, such as the sulfonamides. Hemoglobins Bibba, Torino, Hammersmith, Sydney, Koln, and Kansas are also unstable and involve changes in the normal heme-globin relations. 3 :> When hemolysates containing these hemoglobins are heated to 50° C., the abnormal hemoglobin is precipitated.

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Table 1. Abnormal Hemoglobins of Clinical Significance':' NAME

SUBSTITUTION

CLINICAL FINDINGS

Bibba Bronx-Riverdale C J. Capetown Chesapeake Freiburg Genova Gun Hill Hammersmith Kansas Kempsey Koln M Boston M Iwate M Saskatoon M Milwaukee M Hyde Park Philly Rainer Sydney S (sickle) Torino Wien Yakima Zurich

(, 136 leu ~ pro {3 24 gly .... arg {36 glu ~ lys ()( 92 arg ~ glu (, 92 arg .... leu {3 23 val .... 6 {3 28 leu --,> pro {3 91 to 97 .... 0 {3 42 phe .... ser {3 102 asn .... thr {3 99 asp .... his {3 98 val --,> met (, 58 his .... tyr " 87 his --,> tyr {3 63 his --,> tyr {3 67 val --,> glu {3 92 his .... tyr {3 35 tyr ..... phe {3 145 tyr --,> his {3 67 val --,> ala {3 6 glu --,> val ,,43 phe .... val {3 130 tyr --,> asp {3 99 asp --,> his {3 63 his --,> arg

Inclusion body anemia, unstable 50 C. Inclusion body anemia, unstable 50 C. Target cells, hemolytic anemia Mild polycythemia, high 0, affinity Polycythemia, high 0, affinity Cyanosis, high 0, affinity Inclusion bodies, hemolytic anemia, labile at 50° C. Hemolytic anemia Inclusion bodies, unstable 50° C. Cyanosis, unstable 50" C. Polycythemia, high 0, affinity Hemolytic anemia Cyanosis, methemoglobinemia Cyanosis, methemoglobinemia Cyanosis, methemoglobinemia Cyanosis, methemoglobinemia Cyanosis, methemoglobinemia Mild hemolytic anemia Polycythemia, high 0, affinity Hemolytic anemia, unstable 50° C. Hemolytic anemia, sickling Inclusion bodies, unstable 50" C. Hemolytic anemia, unstable 50° C. Polycythemia, high 0, affinity Hemolytic anemia with drugs 0

C

':'Summarized from reference 35 (partial list).

In another group of well-characterized hemoglobins, the M he moglobins, the normal heme-globin relationships are also abnormal and the heme iron is stabilized in the ferric state and results in methemoglobinemia and cyanosis,'''' In hemoglobin S (sickle cell hemoglobin), the substitution of a valine for a glutamic acid in the sixth position of the f3 chain results in its peculiar insolubility in the deoxygenated state and the characteristic sickling of the cells with its severe clinical sequelae. In another group of abnormal hemoglobins, exemplified by hemoglobin Chesapeake, the amino acid substitution leads to an increased affinity of the hemoglobin for oxygen and less release of oxygen to the tissues, resulting in anoxia and secondary polycythemia. Hemoglobins of this type are associated with amino acid substitutions at contacts between the ex and f3 globin chains.'" Most of the abnormal hemoglobins differ in charge from hemoglobin A and can be separated by electrophoretic techniques, although a few, including hemoglobins Hammersmith, Wien, Torino, and Genova, were discovered by their instability at 50° C.'l' The continuing discovery of new hemoglobin mutants and an analysis of their properties is permitting a detailed evaluation of the sites in hemoglobin which are vital to normal function. The abnormal he moglobins described to date which are clinically significant are listed in Table 1.

Genetics of Normal Hemoglobins Each individual inherits two genes for each globin chain, one from each parent. The allelism of (~ and f3 chain genes is reinforced by the

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finding that no subject with the possibility of inheriting more than two genes for a single globin chain has done so to date. For example, if one parent carries the {3 chain genes for Wand W as in patients with S-C disease, while the other has only normal hemoglobin A genes, the offspring will have either hemoglobins S and A or C and A, but no individual will produce hemoglobins S, C, and A. An individual inheriting one gene for an abnormal chain and one for normal hemoglobin A (i.e., an S-A patient) is said to be heterozygous for the abnormal hemoglobin; subjects who inherit two abnormal genes for a given globin chain (i.e., S-S patients) are called homozygotes. Although the presence of only two genes for 0', {3, and 0 chains appears established, the number of genes for y chains is less clearly defined. Recently, two y chains differing by only a single amino acid have been found in variable proportions in the peripheral blood of several normal patients. The marked variation in the amount of each of these y chains is consistent with the presence of either several closely linked y chain genes within a single genetic locus, or several separate genes for y chains inherited independently.39

Proportions of Different Hemoglobins in Heterozygotes The amount of an abnormal globin chain present in the peripheral blood of patients heterozygous for an abnormal hemoglobin (i.e., S-A) varies considerably. In most cases, hemoglobin A is present as more than 50 per cent of the total hemoglobin. In patients heterozygous for hemoglobin Zurich, only 30 per cent of the total hemoglobin is hemoglobin Zurich, while the rest is hemoglobin A. In contrast, in sickle cell heterozygotes, between 35 and 45 per cent of the total hemoglobin is hemoglobin S, while the remainder is hemoglobin A. Two alternatives have been suggested to explain these findings: (1) the cells containing the abnormal hemoglobins may have a shorter life span in the peripheral circulation, which is particularly likely in the case of unstable hemoglobins such as hemoglobins KOln and Zurich; 26, 37 (2) there may be a lower rate of production of the abnormal globin chains compared to that of hemoglobin A.

INTRACELLULAR MECHANISMS REGULATING GLOBIN CHAIN SYNTHESIS The factors which determine the type of hemoglobin produced by a particular cell or cell lineage are unknown. The role of changes in cell populations in this regard is also unclear at present. In mouse embryonic development, three specific embryonic hemoglobins are produced in the yolk sac until day 11 of fetal life; subsequently, the synthesis of another hemoglobin, adult hemoglobin, begins in liver erythroid cells. 28 ,33 No such organ-specific hemoglobin production has been documented, however, during fetal or adult human development. The factors responsible for the change from hemoglobin F to hemoglobin A production during human fetal development are unknown, although it appears that single cells can produce both of these hemoglobins. 34 Little is known of the detailed structure of the genes for individual globin chains, although genetic data indicate that the 0', {3, and y loci

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are not closely linked, while the f3 and 0 chain loci probably are. Hemoglobin Lepore may result from a crossover mutation between the genes for 0 and f3 chains, since its structure is identical with that of the 0 chain at the N-terminal end and the f3 chain at the C-terminaP The factors controlling the different rates of synthesis of different globin chains are also unknown. The synthetic time for the 0 chains, for example, has been demonstrated to be about one fortieth of that of f3 chains. 36 ,43 The presence of regulatory genes, which might control the activity of the globin structural genes, has not been documented in mammalian cells, although such genes regulate enzyme synthesis in bac teria. 25 The synthesis of the globins occurs predominantly on the ribosomes in the cytoplasm, or, more specifically, the polyribosomes, which are a complex of two or more ribosomes (Fig. 1). A single ribosome of erythroid cells has a diameter of about 250 angstroms and a coefficient of sedimentation of 80S, and is composed of approximately 50 per cent protein and 50 per cent ribonucleic acid (RNA), which is referred to as ribosomal RNA. Messenger RNA is the form in which information contained in deoxyribonucleic acid (DNA) is delivered to the site of protein synthesis, the polyribosomes. In erythroid cells, messenger RNA is relatively stable. 32 The capacity for protein synthesis persists through the reticulocyte stage, although RNA synthesis occurs predominantly before the polychromatophilic erythroblast stage and no RNA is formed in reticulocytes.

Figure 1. Election micrograph of a section of a reticulocyte demonstrating mitochondria (m) and numerous polyribosomes (r). x 35,000. (Courtesy of Dr. Richard A. Rifkind.)

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Amino acids must be "activated" for assembly into polypeptides. This requires energy, specific enzymes and another class of RNA molecules called transfer RNA. A particular transfer RNA can accept only a specific amino acid, although there are several amino acids for which there is more than one particular transfer RNA. The amino-acyl-transfer RNA complex attaches to the ribosomes in a sequence determined by base pairing between the anticodon triplet of bases of transfer RNA and the codon triplet of bases of the messenger RNA associated with ribosomes. The detailed mechanisms involved in initiation of polypeptide chain, transfer of an amino acid to the growing end of the polypeptide chain, and release from the ribosomes of the completed protein are not well understood.

THE THALASSEMIA SYNDROMES The thalassemia syndromes are a group of genetically determined anemias in which there is decreased or absent production of one type of structurally normal globin chain.:1!' 41 The most common types of thalassemias are the ex and 13 thalassemias; although isolated reports of absent y chain production exist, this entity is poorly defined.

f3 Thalassemia THE HETEROZYGOUS STATE. The heterozygote for 13 thalassemia inherits one gene which results in normal f3A production, and a 13 thalassemia gene which leads to either decreased or absent 13 chain synthesis. No structurally abnormal 13 chains have thus far been identified in the few patients with 13 thalassemia in whom peptide analysis of chains has been performed. 1H Genetic evidence that the defect in thalassemia selectively affects 13'\ chain synthesis was first derived from the observation that patients with sickle cell trait and 13 thalassemia trait have more hemoglobin S than hemoglobin A, a reversal of the ratio of amounts of the two hemoglobins in patients with sickle cell trait. This preferential effect of the 13 thalassemia gene on normal 13 1 chain production is known as interaction. In addition, the observation that (j and y chain synthesis are either normal or increased in patients with 13 thalassemia also indicates a specific defect in 13 chain synthesis (Table 2). Clinically, most patients with 13 thalassemia trait or thalassemia minor are asymptomatic, although they may have splenomegaly and a mild anemia. The peripheral blood smear characteristically shows hypochromic, microcytic cells with some target cells. In the vast majority of cases, the diagnosis is confirmed by the finding of an elevation in the level of hemoglobin Az to above 2.5% (Table 2). In a few families, the hemoglobin Az level is normal and the hemoglobin F level is elevated, while rarely both hemoglobin Az and hemoglobin F levels are elevated. THE HOMOZYGOUS STATE. The homozygous state of 13 thalassemia, thalassemia major or Cooley's anemia, is a severe disease which is usually lethal during the first two decades of life. The patients have significant bone changes and hepatosplenomegaly, and a severe anemia which usually requires frequent blood transfusions. The peripheral

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GENETIC CONTROL O~" HEMOGLOBIN SYNTHESIS AND THALASSEMIA

Table 2.

Percentage of Different Hernoglohins in Thalassernias A

A,

normal normal 10-90 < 50

> 2.5 normal normal > 2.5

normal > 2.0 10-90 normal > 50% (HbS) or high

normal 70-95

normal normal orlow 0

normal normal

F

OTHER

f3 Thl1ll1ssemia

Trait or Minor (heterozygote) High A, (common) High F (uncommon) Major (homozygote) Sickle-f3 thalassemia

et Thalassemia

Trait (heterozygote) Hemoglobin H disease Homozygote

0

5-30% (HbH) 100% (Hb Barts)

0

smear shows marked variation in the size and shape of cells, with many hypochromic cells and target cells. The mortality from hemochromatosis secondary to blood transfusions is high. HEMOGLOBIN SYNTHESIS IN f3 THALASSEMIA. In thalassemia major, the total amount of hemoglobin A production is reduced to a variable extent, resulting from the presence of two thalassemia genes. It is now recognized that there is marked heterogeneity in the amount of f3 chain production in different subjects with thalassemia major (Table 3).",1",41 In some cases, no f3A is produced and thus no hemoglobin A is present, while in others, significant amounts of hemoglobin A can be demonstrated. The bulk of the hemoglobin in these patients is hemoglobin F, but the degree of compensation by hemoglobin F for the lack of adequate hemoglobin A synthesis is incomplete. It has been shown that the cells containing the most hemoglobin F survive longest in the peripheral circulation. 17 The lack of complete compensation by 'Y chains for the Table 3.

Synthetic Rates in Thalassernia etl f3

Normals

f3 Thall1ssemia'

Trait or minor Sickle thalassemia Thalassemia major With high f3 synthesis With low f3 synthesis With no f3 synthesis

a Thalassemia 1I, 27

Trait HbH disease Homozygote

RATIO':'

0.9-1.1 2.0-2.5 2.0-2.5 4.8

15.0

0.7-0.8 0.3-0.6

"Relative rates of synthesis of et and f3 chains (alf3 ratio) by reticulocytes incubated with radioactive amino acids.

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reduction in (3 chain synthesis is somewhat curious when one considers another inherited disorder, hereditary persistence of hemoglobin F. The patients homozygous for this condition produce no hemoglobin A or A2 at all and yet are not anemic. 1:l This indicates complete compensation by )' chain synthesis for the lack of (3 chain. Perhaps the presence of 0 and (3 chain production limits)' chain compensation in the cells of patients with thalassemia major. The deficiency in (3 chain synthesis in patients with (3 thalassemia can be demonstrated in vitro by incubating their peripheral blood with radioactive amino acids and measuring speCific globin chain synthesis. (3 thalassemia heterozygotes can be readily identified by a characteristic increase in the relative amount of a chain synthesis compared to that of (3 chains (11'/(3 ratio) (Table 3).7 Normally, the production of a and (3 chains is approximately equal, while in (3 thalassemia heterozygotes the 11'/(3 ratio is 2.0 to 2.5. 7 In patients with thalassemia major, there is a marked excess of a chain synthesis relative to that of (3 chains - usually greater than 5 to l.7.42 In addition, several groups of patients with thalassemia major can be distinguished by differences in their ratios of a and (3 chain synthesis (Table 3).5 One group in which the ratio averages 4.8 may be designated as high (3 producers; another group with an 11'/(3 ratio of 15 may be called low (3 producers, while a third group produces no (3 chains at alP THE FATE OF THE EXCESS a CHAINS. The absolute amount of a chains produced in the cells of (3 thalassemic subjects is not significantly different from that of normals. 6 This indicates that normal a chain synthesis in thalassemic cells does not require a normal rate of (3 chain synthesis. The relative excess of a chains produced in these cells is not present in hemoglobins A or F. Instead they form a chain aggregates which conform to no single molecular configuration. 3 , 20 This has been demonstrated by separating newly synthesized hemoglobin from a radioactive thalassemic hemolysate by column chromatography and showing that a chain radioactivity is present as monomers, dimers, and trimers as well as tetramers.:l In addition, if hemoglobin H ((34) is added to a freshly labeled thalassemic hemolysate, all of the excess of a chains become associated with hemoglobin A.3 These experiments suggest that the a chains synthesized in (3 thalassemic cells are structurally normal and aggregate only because of the lack of (3 or )' chains with which to associate. Ultimately, the excess a chains either are destroyed or are precipitated as part of the inclusion bodies found in the cells of patients with this disorder. 14 a chain peptides have been found to predominate in peptide analysis of the inclusions. 16 The presence of these inclusion bodies may lead to selective destruction of the cells containing them and may explain the hemolytic component of the anemia in patients with (3 thalassemia. The marked increase in the number of circulating cells with inclusion bodies after splenectomy supports this concept. Recently it has been shown that radioactively labeled intact cells from thalassemic subjects lose close to 50 per cent of their a chain radioactivity on overnight incubation at 37° C.8 This suggests that degradation as well as preCipitation of a chains may occur in thalassemic cells.

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THE UNDERLYING DEFECT IN f3 CHAIN SYNTHESIS. It has been suggested that f3 thalassemia is due to a primary defect in heme synthesis and that the depression in hemoglobin formation is secondary." There is a defect in he me metabolism in erythroid cells of patients with thalassemia, but this seems to be secondary rather than primary. Heme is required for the synthesis of all types of hemoglobin, and it is unlikely that a primary defect in heme synthesis could account for the selective depression in one type of globin chain formation. Alternatively, it has been suggested that the thalassemia gene decreases the rate of synthesis of a structurally normal globin chain. There are several mechanisms which could lead to such a decreased rate of synthesis of a structurally normal chain: (1) the amount of messenger RNA for a particular globin chain could be decreased; (2) the messenger RNA for a specific globin chain could be altered so that translation at the ribosome level would be slowed and structurally normal globin formed at a decreased rate; (3) a net depression in globin synthesis might also result from an increased rate of destruction of the messenger RNA ribosome complex directing f3 chain synthesis; (4) the net synthesis of f3 chain would also be decreased if a specific transfer RNA or other factor that is rate-limited to initiation, growth, or release of a given polypeptide chain were altered or deficient. For example, Itano has suggested that an altered nucleotide sequence in the mRNA for thalassemic f3 chains may limit f3 chain peptide assembly by requiring a tRNA which is present in short supply.24 Recent data demonstrate that the time required for f3 chain synthesis in thalassemic cells is similar to that of normal f3 chains. 12 This suggests that thalassemic f3 chain assembly may be normal, although it does not rule out the possibility that diminished f3 chain synthesis results from an abnormality which decreases the rate of f3 chain initiation. In vitro studies have been attempted to clarify the nature of the underlying defect in f3 chain synthesis. To test the possibility that the defect in the thalassemic cell involved the ribosome fraction, a cell-free system was prepared from human erythroid cells and the efficiency with which ribosomes from thalassemic and nonthalassemic subjects supported the synthesis of new protein was compared. 4 Employing such a cell-free system, the ribosomes prepared from cells of thalassemic subjects had a markedly diminished capacity to synthesize protein as compared to that of ribosomes from cells of nonthalassemic subjects. This defect in the activity of ribosomes from cells of thalassemic subjects was not corrected in the cell-free system by the addition of supernatant fractions prepared from cells of nonthalassemic subjects or from rabbit reticulocytes. 4 The polyribosome fraction includes both ribosomes and messenger RNA. The question of which of these two components was responsible for decreased globin synthesis in thalassemia was approached by measuring the response of the ribosomes themselves to an exogenously added synthetic messenger RNA, such as polyuridylic acid. Ribosomes prepared from nonthalassemic and thalassemic cells were comparable in their capacity to respond to polyuridylic-aciddirected phenylalanine incorporation. 4 These data suggested that the

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ribosomes per se were not defective in thalassemic cells; rather, there was some alteration in the messenger RNA in the f3 thalassemic cell. This alteration might be either a decrease in the quantity of or a qualitative alteration in the messenger RNA; either could account for the decreased rate of f3 chain synthesis. It should be emphasized that there is no good evidence that the action of polyuridylic acid accurately reflects that of endogenous or natural messenger RNA.

a Thalassemia THE HETEROZYGOUS STATE. There appear to be at least two types of a thalassemia heterozygotes. 4o They are most easily identifiable at birth when the deficiency of a chains results in increased amounts of hemoglobin Barts (y4).40 Normally, less than 1 per cent hemoglobin Barts is present at birth. One group of a thalassemia heterozygotes has 1 to 2 per cent hemoglobin Barts, while another has more than 5 per cent. The number of patients with 1 to 2 per cent Barts at birth correlates well with the expected frequency of the so-called silent carrier a thalassemia gene. 40 Patients with this gene are clinically and hematologically normal. The subjects with more than 5 per cent hemoglobin Barts at birth are presumably related to adults with so-called a thalassemia trait. These individuals usually have hypochromic blood smears and mild anemia but no characteristic electrophoretic abnormalities; they demonstrate the phenomenon of interaction in association with patients with a chain mutations indicating decreased synthesis of normal a chains. Hemoglobin H (f34) disease appears to be an intermediate state of a thalassemia. It is characterized by splenomegaly, a mild to moderate anemia usually appearing in childhood, and a typical hypochromic, microcytic smear. Hemoglobin H is readily identified in fresh hemolysates of affected individuals as a rapidly migrating hemoglobin on electrophoresis. Usually, 5 to 30 per cent of the total hemoglobin is hemoglobin H. It has been shown that hemoglobin H is unstable, precipitates intracellularly, and leads to the preferential destruction of cells containing it.:IH In the laboratory, incubation of the patient's blood of 37° C. with brilliant cresyl violet results in precipitates and confirms the presence of an unstable hemoglobin. The genetics of hemoglobin H disease remain somewhat unclear. The frequency of hemoglobin H disease is roughly the same as would be expected if both a silent carrier gene from one parent and the gene for a thalassemia trait from the other were requiredP However, there are many cases of hemoglobin H disease in which only one parent is abnormal, and it remains to be proved that the other parent does indeed harbor a silent carrier gene for a thalassemia. THE HOMOZYGOUS STATE. Hemoglobin H disease may be a mild form of the homozygous state of a thalassemia. A complete lack of a chain synthesis in the most severe homozygous state is incompatible with life; the fetus is born dead with hydrops fetalisYo Several such cases have been reported with the only hemoglobin in the fetus being hemoglobin Barts.30 The genetic interrelationship between this lethal

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disease, hemoglobin H disease, and a thalassemia trait remains unclear. HEMOGLOBIN SYNTHESIS IN a THALASSEMIA. In patients with hemoglobin H disease, there is a marked decrease in the ratios of synthesis of a chains relative to that of f3 chains (alf3 ratios), averaging 0.3 to 0.6 (Table 3).0. 27 In patients with a thalassemia trait, these ratios are still abnormal, averaging 0.7 to 0.8. 27 In patients with a presumptive diagnosis of the silent carrier state, the alf3 ratios are close to normal, although they may be slightly below normaP7

SUMMARY At different stages of mammalian development, different he moglobins are produced. Intracellular control of globin synthesis operates both at the level of DNA in the nucleus, and at the polyribosomal site in the cytoplasm. The synthesis of each globin chain is controlled by two genes. In the thalassemia syndromes, there is a quantitative decrease in the production of either a or f3 chains by one or both of these genes. In f3 thalassemia, this results in either decreased or absent hemoglobin A synthesis, with elevated levels of hemoglobin A2 in thalassemia minor and elevated hemoglobin F in thalassemia major. In a thalassemia, the deficiency of a chains leads to excess hemoglobin Barts (y4) at birth, and hemoglobin H (f34) in affected adults. Homozygous a thalassemia is lethal in utero and is characterized by hydrops fetalis.

REFERENCES 1. Baglioni, c.: Correlations between genetics and chemistry of human hemoglobins. In Taylor, J. H., ed.: Molecular Genetics. New York, Academic Press, 1963, Vo!. 1, p. 405. 2. Baglioni, C.: The fusion of two peptide chains in hemoglobin Lepore and its interpretation as a genetic deletion. Proc. Nat. Acad. Sci., 48: 1880, 1962. 3. Bank, A.: Hemoglobin synthesis in f3 thalassemia; the properties of the free" chains. ]. CHn. Invest., 47:860, 1968. 4. Bank, A., and Marks, P. A.: Protein synthesis in a cell-free human reticulocyte system; ribosome function in thalassemia. J. Clin. Invest., 45:330,1966. 5. Bank, A., Braverman, A. S., and Marks, P. A.: Globin chain synthesis in thalassemia. Ann. N.Y. Acad. Sci., in press. 6. Bank, A., Braverman, A. S., O'Donnell, J. V., and Marks, P. A.: Absolute rates of globin chain synthesis in thalassemia. Blood, 31 :226-233, 1968. 7. Bank, A., and Marks, P. A.: Excess of" chain synthesis relative to f3 chain synthesis in thalassemia major and minor. Nature, 212:1198, 1966. 8. Bank, A., and O'Donnell, J. V.: Intracellular proteolysis of free a chains in thalassemia. Chn. Res., 16:533, 1968. 9. Bannerman, R. M.: Thalassemia: A Survey of Some Aspects. New York, Grune & Stratton, 1961. 10. Braunitzer, G.: Phylogenetic variation in the primary structure of hemoglobins. J. Cel!. Phys., 67, Supp!. 1, 1966, p. 1. 11. Clegg, J. B., and Weatherall, D. J.: Hemoglobin synthesis in thalassemia (hemoglobin H disease). Nature, 215:1241,1967. 12. Clegg, J. B., Weatherall, D. J., Na-Nakorn, S., and Wasi, P.: Hemoglobin synthesis in f3 thalassemia. Nature, 220:664, 1968. 13. Conley, C. C., Weatherall, D. J., Richardsen, S. N., Shepherd, M. D., and Charache, C.: Hereditary persistence of fetal hemoglobin: Study of 79 affected persons in 15 Negro families in Baltimore. Blood, 21 :261, 1963. 14. Fessas, P.: Inclusions of hemoglobin erythroblasts and erythrocytes of thalassemia. Blood, 21 :21, 1963. 15. Fessas, P.: The heterogeneity of thalassemia. Plenary session papers, XII Congress, Int. Soc. Hemat., 1968, p. 52. 16. Fessas, P., and KaJtsoya, A.: Peptide analysis of the inclusions of erythroid cells in f3 thalassemia. Biochem. Biophys. Acta, 124:430, 1966.

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