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CHAPTER 48 HEMOGLOBINOPATHIES ASSOCIATED WITH UNSTABLE HEMOGLOBIN

CHAPTER 48 HEMOGLOBINOPATHIES ASSOCIATED WITH UNSTABLE HEMOGLOBIN
Williams Hematology

CHAPTER 48 HEMOGLOBINOPATHIES ASSOCIATED WITH UNSTABLE HEMOGLOBIN

ERNEST BEUTLER

Definition and History
Etiology and Pathogenesis

Mode of Inheritance
Clinical Features
Laboratory Features
Differential Diagnosis
Treatment, Course, and Prognosis
Chapter References

Mutations that cause destabilization of the hemoglobin tetramer are an uncommon cause of hemolytic anemia. In contrast to the hemolytic anemias caused by enzyme deficiencies, a dominant mode of inheritance characterizes the unstable hemoglobins. Heinz bodies are a characteristic feature of the red cells in the blood when splenectomy has been carried out. Hemolytic anemia may be precipitated by the ingestion of oxidative drugs. The diagnosis is established by precipitating the unstable hemoglobin in a system in which the hemolysate is heated or incubated in a mixture of isopropanol and buffer. Although splenectomy has occasionally ameliorated the anemia, it should be avoided in most cases, because it has sometimes been followed by fatal thromboembolic complications.

DEFINITION AND HISTORY
The sporadic occurrence of hemolytic anemia with the appearance of inclusion bodies in the red cells was occasionally observed in the 1940s and 1950s,1,2 and 3 but it was not until 19624,5 that it was recognized that such patients had abnormal hemoglobins that spontaneously denatured within the circulating red cell. The unstable hemoglobins that will be discussed in this chapter are those that result from a mutation that changes the amino acid sequence of one of the globin chains. Homotetramers of normal b chains (hemoglobin H) or normal g chains (hemoglobin Barts) are also unstable hemoglobins. These unstable hemoglobins occur in patients with a thalassemia and are discussed in Chap. 46. Hyperunstable hemoglobins6 have defects that are so severe that the globin chain is not found in the red cells, but their formation can be deduced from the DNA sequence.
ETIOLOGY AND PATHOGENESIS
The tetrameric hemoglobin molecule has evolved so that a variety of noncovalent forces maintain the structure of each subunit and bind the subunits to each other. The delicate balance that allows the molecule to change from one state to another, facilitating its oxygen-binding function while maintaining its structural integrity, has been discussed in Chap. 28. It is not surprising that a variety of amino acid substitutions or deletions will weaken the forces that maintain the structure of hemoglobin. When this occurs, the hemoglobin molecule denatures and precipitates as insoluble globins. These precipitates often attach to the cell membrane and are recognized as Heinz bodies.
Instability of hemoglobin can arise from any one of the following processes:

1.
Replacement of an amino acid that contacts the heme group or produces a change in the property of the heme pocket often results in an unstable molecule with a tendency to lose heme from the abnormal globin chains. HbHammersmith,7,8 HbSendagi, HbAlesha,10 and HbLa Roche-sur-Yon11 are examples of this type of unstable hemoglobin.

2.
Replacement of nonpolar by polar residues at the interior of the molecule results in gross distortion of the protein, particularly if the new polar residue remains in the interior portion of the molecule, as in HbBristol12 and HbVolga.13

3.
Deletions or insertions of additional amino acids, particularly when critical helical regions of the sequence are involved, creates instability, as in HbNiteroi14 and HbMontreal.15

4.
Replacements at intersubunit contacts, particularly those between the a1 and b1 chain, create instability so that dissociation into monomers may occur. HbPhilly16 and HbTacoma17 are mildly unstable for this reason. Replacements at the contact between the a1 and b2 globin monomers usually result in hemoglobins with a high oxygen affinity.

5.
If proline is introduced into an a helix beyond the third residue, distortion of the helix results in instability.18 Variants in which proline substitution results in instability include HbDuarte19 and HbSanta Ana.20

6.
In areas of the hemoglobin molecule in which atoms are very tightly packed, substitution of amino acids with larger side chains for glycine may produce marked changes in stability. In particular, at the points where the B and E helices approach each other there is no room for the substitution of larger amino acids for glycine at B6 and E8. HbRiverdale-Bronx,21 HbSavannah,22 and HbMoscva23 arise in such a fashion.

7.
Replacement of a hydrophobic residue that normally fits into a hydrophobic pocket with a more hydrophilic amino acid, such as the substitution of histidine for leucine at b81 in HbLa Roche-sur-Yon.11
Many unstable hemoglobins have an increased susceptibility to oxidation to methemoglobin. However, the exact sequence of events that leads to the precipitation of hemoglobin is not fully understood and very likely varies with different unstable hemoglobins. The formation of hemichromes may be involved. These are compounds in which heme has been removed from its normal binding site and has become bonded to another part of the globin molecule.24 These pigments can be shown to form during in vitro denaturation of some abnormal hemoglobins,25 and they are present in hemoglobin H inclusion bodies.26 The release of activated oxygen in the form of superoxide radicals with the subsequent formation of peroxide and the hydroxyl radicals27,28 may also play a role. The attachment of Heinz bodies to the cell membrane impairs the deformability of the erythrocyte and impedes its ability to negotiate the narrow spaces between the endothelial cells lining the splenic sinuses. The “pitting” of Heinz bodies from the erythrocyte results in loss of membrane and ultimately in destruction of the red cells. Although Heinz bodies are formed, their presence in the blood does not become a prominent feature except in patients who have been splenectomized (see “Laboratory Features”). Selected unstable hemoglobins that have been characterized are listed in Table 48-1. Detailed tabulations are available.29

TABLE 48-1 THE UNSTABLE HEMOGLOBINS

Hyperunstable hemoglobins are characterized by b-globin formation that is so defective that no b-chains are found. However, they differ from the b-thalassemias in that inheritance is dominant, i.e., a single copy of the mutant gene is all that is required to give the clinical phenotype. They may be due to single base substitution, deletion of codons, frameshifts leading to elonged b-chains, or premature terminations.30
MODE OF INHERITANCE
Unstable hemoglobins are generally inherited as autosomal dominant disorders. Affected individuals are usually heterozygotes who have inherited the defect from one of their parents and who on the average will transmit it to one-half of their offspring. Since unstable hemoglobins produce a disease state, genes for these disorders are subjected to negative selection, and the continued existence of the unstable hemoglobinopathies in the population is the result of such new mutations. Thus, occasionally patients with an unstable hemoglobin are encountered neither of whose parents had the abnormality. The homozygous state for the unstable hemoglobins HbSun Prairie31 and HbBushwick32 have been observed, and a homozygous-like state can occur when an unstable b chain mutation is inherited together with a bo thalassemic gene.19,33
Over 80 percent of unstable hemoglobins that have been characterized affect the b chain. This probably reflects the fact that the normal genome contains four copies of the a chain. The clinical effects of such mutants, affecting only one-fourth of the total hemoglobin formed, is apt to be less pronounced than those of b-chain mutants, in which one-half of the hemoglobin produced is abnormal. Thus, many a-globin mutations are likely to be overlooked.
Although most patients with unstable hemoglobins have been found to have a combination of hemoglobin A and the unstable hemoglobin in their red cells, there are a number of reports of the inheritance of unstable hemoglobins with other hemoglobinopathies.19,34,35,36,37 and 38
CLINICAL FEATURES
A broad spectrum of clinical manifestations can be induced by unstable hemoglobins. In most cases, hemolysis is well compensated, and some hemoglobins that are unstable in vitro (e.g., HbMuscat39) are not associated with hemolysis at all. When an unstable hemoglobin also has a left-shifted oxygen dissociation curve, i.e., a raised O2 affinity, the hemoglobin level may be in the upper portion of the normal range. Episodes of infection and treatment with “oxidant” drugs are likely to precipitate hemolytic episodes in persons whose anemia is well compensated under ordinary circumstances. It is at this juncture that the diagnosis is often first made. In the case of patients who have particularly unstable variants, such as HbHammersmith,7 HbBristol,12 HbSanta Ana,20 or HbMadrid,40 a chronic hemolytic anemia may become evident during the first year of life as g chain production is replaced by production of the mutant b chain. In contrast, in the rare instances where the g chain bears the abnormality,41 the hemolytic anemia is evident at birth, and it disappears as normal b chains are formed.
Physical findings include jaundice, splenomegaly, and, when the anemia is severe, pallor. In some patients, dark urine has been observed, probably as a result of the excretion of dipyrrole pigments derived from the catabolism of free heme groups or of Heinz bodies.42 In some instances methemoglobulinemia may develop, and cyanosis may then be evident.
LABORATORY FEATURES
The hemoglobin concentration of the blood may be normal or decreased. The mean corpuscular hemoglobin is usually diminished because of the loss of hemoglobin from the red cells as a result of its denaturation and subsequent pitting from the erythrocytes. The blood film may show slight hypochromia, and, in addition, poikilocytosis, polychromasia, anisocytosis, and some basophilic stippling may be evident. Hyperunstable hemoglobins, in particular, are associated with severe hypochromia of the erythrocytes and present clinically as dominant b-thalassemia. Reticulocytosis is often out of proportion to the severity of the anemia, particularly when the abnormal hemoglobin has a high oxygen affinity. After splenectomy many Heinz bodies may be found in the circulation. Hemoglobin F levels may be increased.43
Diagnosis of this disorder usually depends upon the demonstration of the presence of an unstable hemoglobin. Three tests are used for this purpose. The most convenient is the isopropanol stability test.44 The heat stability test is also useful45 but is somewhat more difficult to interpret. It has been found, however, that at least one unstable hemoglobin, hemoglobin Olmsted, can be detected by heat stability but not isopropanol stability.46 Finally, incubation of blood with brilliant cresyl blue generates Heinz bodies in hemoglobin H disease.47,48 Further identification of unstable hemoglobins is aided by procedures such as hemoglobin electrophoresis; however, the electrophoretic pattern is often normal, and the diagnosis of the hemoglobinopathy cannot be ruled out in this way. The oxygen affinity of unstable hemoglobins is often altered, and the determination of the P50 may help in detecting and characterizing the unstable hemoglobin. In the final analysis unstable hemoglobins can be identified only by DNA analysis10,43,49,50 or by physical separation of the abnormal hemoglobin from the normal hemoglobin, followed by globin chain separation and peptide analysis.
DIFFERENTIAL DIAGNOSIS
The possibility that an unstable hemoglobin is present should be considered in all patients who present with the clinical picture of hereditary nonspherocytic hemolytic anemia (Chap. 45), particularly when hypochromia of the red cells is present and when the extent of the reticulocytosis is out of keeping with the degree of anemia. Not all patients with a positive test for unstable hemoglobins should be classified as having this disorder. The stability of methemoglobin, hemoglobin F, and sickle hemoglobin is appreciably less than that of hemoglobin A, and false-positive isopropanol stability tests may be obtained in patients with increased quantities of these hemoglobins. Hemoglobin H (b4) and hemoglobin Barts (g4) are unstable. These fast-moving hemoglobins can be detected on electrophoresis. Patients whose red cells contain these hemoglobins are diagnosed as having a thalassemia (see Chap. 46).
Sometimes the hemoglobins are so unstable that none of the protein can be detected. Such abnormal hemoglobins have been diagnosed by DNA-based analysis.10,43,49,50
TREATMENT, COURSE, AND PROGNOSIS
Most patients with unstable hemoglobins follow a relatively benign course. As with other hemolytic states, gallstones are common, and cholecystectomy may be required. Hemolytic episodes may be precipitated by infection or by the ingestion of “oxidative” drugs. Sulfonamides have been particularly prominent in inducing hemolysis, and derivatives that do not produce hemolysis in G-6-PD deficiency have been shown to precipitate hemolysis in patients with some unstable hemoglobins. A few deaths that are believed to have been directly related to unstable hemoglobins have been reported. A patient with HbHirosaki is thought to have died following a hemolytic crisis precipitated by a common cold.51 Two sisters with HbDuarte19 died of thromboembolic complications less than a year following splenectomy. This unstable variant has an increased oxygen affinity, and it is likely that a combination of postsplenectomy erythrocytosis and thrombocytosis led to the demise of the patients.
Treatment is not usually required. As in the case of other hemolytic disorders, folic acid in a dose of 1 mg per day is often given, but its usefulness has not been established. “Oxidant” drugs such as those listed in Table 45-5 should be avoided. In addition, the use of all sulfonamides should be eschewed, particularly in the case of those variants which have been associated with drug-induced hemolysis. Splenectomy has proved to be useful in some patients with splenomegaly and severe hemolysis,52,53 while others have enjoyed little benefit.46 In view of the fact that patients with high-oxygen-affinity unstable hemoglobin have died after a splenectomy19 and that thromboembolic complications have been reported in a number of other patients,54 it is probably best to avoid splenectomy. Preliminary results suggested that hydroxyurea therapy might be useful,53 presumably by increasing the level of fetal hemoglobin.
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19.
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20.
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21.
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22.
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23.
Idelson LI, Didkowsky NA, Casey R, Lorkin PA, Lehmann H: New unstable haemoglobin Hb Moscva, beta 24(B6) Gly®Asp found in the U.S.S.R. Nature 249:768, 1974.

24.
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25.
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26.
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27.
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28.
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29.
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30.
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31.
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32.
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33.
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34.
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35.
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36.
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37.
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38.
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39.
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40.
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41.
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42.
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43.
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44.
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45.
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46.
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47.
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48.
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49.
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50.
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51.
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52.
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53.
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54.
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Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

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