Homozygous CC individuals have a mild hemolytic anemia which is generally asymptomatic and rarely life-threatening. The anemia is moderate, the MCV is reduced (50-60 fl), there is minimal reticulocytosis and splenomegaly is common. The peripheral blood film is fairly characteristic and, with some training of the observer, diagnostic. The red cells are hypochromic, due to their flatness [rather than low mean corpuscular hemoglobin (MCH) or MCHC; in fact, they have a normal MCH and an increased MCHC]. Folded and target cells are prominent. Red cells may show intracellular tetragonal crystals of Hb C, best detected in reticulocyte smears, where they retain their red color.
Regarding the genetics of this disease, the PC gene (p6 Val^ Lys), a single base substitution, has a frequency one-quarter that of the pS gene among African-Americans. The pC gene most likely originated in Burkina Faso, where we find the highest frequency (up to 50% of the population), decreasing concentrically, and encompassing Mali, Ivory Coast and Ghana, all of which are east of the Niger river.
The pathophysiology of CC disease is dominated by the high tendency of Hb C to produce oxygenated tetragonal crystals (Plate 14.2). The CC red cells are uniformly denser than AA cells. This effect is associated with very high expression of K:Cl co-transport (higher than in SS cells, in spite of lower reticulocyte count), a transport system that, by extruding K+ and water, can dehydrate red cells. There is evidence that the kinetics of this transport is altered in CC cells, with slower than normal turn-off, when the stimulus for activity, volume increase or low pH is removed. Whether this alteration is sufficient to explain the pathophysiology of CC cells and the mechanistic basis for the interaction of Hb C with the membrane remains to be determined.
Recent epidemiological data from Burkina Faso reveals that homozygous CC individuals are particularly resistant to dying of malaria, which suggest that Hb C will eventually supersede the Hb S gene in the populations where the two mutants coexist. This is congruent with the previous findings that Hb CC red cells were not able to release the merozoites as normal red cells or AC red cells.
The multiplication rate of P. falciparum is measurably lower in CC cells than in normal red cells, probably due to the ring forms and trophozoites disintegrating within a subset of CC cells. In addition, the knobs present on the surface of infected CC cells are fewer in number and morphologically aberrant when compared with those on AA cells. It appears that only a subset of CC cells supports normal parasite replication. Hb C will eventually predominate due to the greater protection from malaria afforded to their carriers, who will be less likely to die before the reproductive period and hence will be able to transmit the Hb C gene to their descendants more efficiently than the sickle gene.
Hb C nucleates and grows by the attachment of Hb C molecules from the solution, but concurrent amorphous phases, spherulites, and microfibers are not building blocks for the crystal. Hb C crystallization is possible because of the huge entropy gain, likely stemming from the release of up to 10 water molecules per protein intermolecular con-tact-hydrophobic interaction. The higher crystallization propensity of oxyHb C is attributable to increased hydro-phobicity resulting from the conformational changes that accompany the Hb C 06 mutation. The oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of Hb C, which energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.
The presence of circulating tetragonal crystal-containing red cells in splenectomized CC patients does not result in vaso-occlusion, probably because the CC tetragonal crystals are crystals of oxyHb C, which melt as they approach the capillaries. DeoxyHb C has a different crystal form, but not enough of the large crystals to generate pathology.
SC disease is genotypically a double heterozygote; that is, a combination of sickle trait and Hb C trait. Since neither of these trait forms independently has a phenotype, the clinical picture of SC disease requires an explanation (see below).
SC disease is milder than sickle cell anemia, particularly in the first 20 years of life, in which the mortality approaches zero. Red cell survival is about 27 days compared with 17 days for sickle cell anemia red cells. Nevertheless, three complications are inappropriately severe: osteonecrosis, retinopathy and acute chest syndrome. SC patients frequently retain their spleen in adulthood, and have less anemia, lower MCV, lower reticulocyte count, fewer very dense and dense red cells and lower Hb F levels, on average, than sickle cell anemia patients. Their survival also seems to be better.
Like CC cells, the blood film in SC disease may be diagnostic. In addition to the cells common to CC disease (flat and apparently hypochromic cells), they exhibit target cells, and more specifically a few ISCs, as well as intracellular tetragonal crystals, better seen in reticulocyte smears (Plate 14.3).
The pathophysiology of SC red cells derives from the contribution that Hb C makes to the red cell: like CC cells (see above), SC cells are denser than AA or most of the sickle cell anemia red cells (Plate 14.4). The increase in MCHC implicit in this effect promotes the polymerization of Hb S, leading to more sickling than expected for a cell with only 50% Hb S. Interestingly, Hb S in turn favors Hb C crystallization; this explains, in addition to the differences in splenic activity, why crystals are generally more prominent in SC disease than in CC. Fortunately, for the reasons expressed above, this situation does not lead to an increase in vaso-occlusion beyond that which is predictable from the solubility of the available Hb S with the MCHC for SC cells.
Homozygous Hb E and Hb E/p thalassemia
The homozygote for Hb E has the phenotype of P thalasse-mia trait because this abnormal hemoglobin, common from the Eastern provinces of India to the Philippines, is both a mutation of the sequence of the P chains and a thalassemia, due to the generation of an alternative splicing site by the mutation.
Populations living near the common border of Cambodia, Laos and Thailand (the Khmer people) have the highest incidence of this abnormal hemoglobin, the selection pressure for which is resistance to infection by P. falciparum malaria.
The clinical syndrome is very mild, with no or minimal anemia (once the nutritional causes of anemia are treated) and low MCV, with target cells in the smear and hypochromia. Density gradients reveal that although the red cells are small they have a normal MCHC, as a consequence of a diminished MCH, due to the thalassemic component of this disease. People who inherit Hb E and P-thalassemia trait present a thalassemia major or intermedia picture (see Chapter 1).
The protective effect of Hb E against P. falciparum malaria was assessed in a mixed erythrocyte invasion assay demonstrating that the parasite preferentially invaded normal red cells compared with abnormal Hb AE, EE, E-P-thalassemia red cells. The heterozygote AE cells were the worst target, with invasion restricted to approximately 25% of the red cells. Hb AE might have an unidentified membrane abnormality that renders the majority of the red cell population relatively resistant to invasion by P. falciparum. This will reduce para-sitemia and hence will reduce the lethality of the infection and is consistent with the Haldane hypothesis of heterozygote protection against severe malaria for Hb E.
Preliminary evidence of iron loading in affected patients with Hb P-thalassemia in Sri Lanka suggests variable but accelerated gastrointestinal iron absorption. The iron loading associated with chronic transfusions in patients with Hb E-P-thalassemia is similar to that observed in patients with P-thalassemia. These data, which represent the only cohort of patients with Hb-P-thalassemia to have undergone quantitative assessment of body iron burden, suggest that that guidance for the assessment of iron loading and initiation of chelating therapy in patients with P-thalassemia may be also applicable to those with Hb E-P-thalassemia. Further quantitative studies in both non-transfused and transfused patients will be necessary to settle this issue definitively.
Dominant sickle mutations refer to P gene mutations that include the PS mutation and exhibit a second mutation in the same chain. This turns the chains into super-Hb S, which produces symptoms in the heterozygote. Two instances have been characterized. Hb S-Antilles (P6 Glu^Val; 23 Val^Ile) is expressed in the heterozygote at about the 40% level and produces a syndrome resembling a mild sickle cell anemia phenotype. The mechanism is complex; the second mutation increases the solubility of deoxyHb S from 17 g/dl to about 11 g/dl, with the result that Hb S polymerizes more readily and more extensively. The second mutation has an oxygen equilibrium effect that favors sickling.
The other dominant Hb S mutation is Hb S-Oman (P6 Glu^Val; 121 Glu^Lys), which generates an even more powerful super-S because, even at expression levels of about 20% (resulting from concomitant —a/—a), it has a phenotype very similar to Hb S-Antilles. Since the two dominant forms of Hb S have the same solubility in the deoxy state, the more severe phenotype present implies that the 121 second mutation (identical to that in Hb Ob) must produce pathology of its own. This hypothesis is confirmed by the hemolytic anemia present in individuals homozygous for Hb Ob or Hb G d,.. . a
There are about 100 mutations that render the hemoglobin molecule unstable (e.g. Hb Köln) but their incidence is low. They are generally inherited in an autosomal dominant manner. Mutations may render hemoglobin unstable and produce a hemolytic syndrome by four mechanisms:
• when the mutation introduces either a bulky or a charged side chain in the interior of this globular protein
• when the mutation introduces, in the a-helix portion of the molecule, a side chain that is not a-helix-friendly
• when the mutation destabilizes the heme attachment to the globin
• when the mutation interferes with the stability of the contact area of atßt, a dimer that normally does not dissociate.
Hb Köln (ß 98VaUMet) is the most common of all the unstable hemoglobins, and affects all ethnic groups. It is difficult to diagnose by electrophoresis because of its instability and indistinct electrophoretic pattern. Two light bands are commonly observed because of the severe instability. The best test is isopropanol solubility, in which the hemolysate turns opaque, and a precipitate can be observed after centrifuga-tion.
The patients, who can be of any ethnic origin, exhibit a mild to moderate hemolytic anemia and the presence of pigmen-turia (mostly dipyrroles). The mechanism of the hemolytic anemia is the intracellular release of the hemes, which leaves very unstable tetramers, the formation of hemochromes and subsequently Heinz bodies, and the recognition of the abnormal red cells with hemoglobin/hemochromes attached to membranes by the spleen and other macrophages. Thrombosis is a recognized complication after splenectomy.
There is no need for treatment except for folic acid supplementation and attention to the possibility of aplastic crises due to parvovirus B19. Rarely, these patients require splenectomy.
The sigmoid curve of the oxygen binding of hemoglobin is the product of the low affinity of the deoxy tetramers (T state) (e.g. Hb Chesapeake), which turn into the R state when two of the four hemes are oxygenated. After this molecular switch the hemoglobin acquires high affinity for oxygen. Mutations that stabilize the R state of the hemoglobin and interfere with the switch to T state will tend to have higher affinity than normal. Hence, the receptor mechanism for hypoxia in the kidney will interpret this situation as evidence of the presence of hypoxia and respond with increased secretion of erythropoietin, and stimulate an increase in the number of red cells and hematocrit.
There are around 100 high-affinity hemoglobins which are, on the whole, rare. The first one described was Hb Chesapeake, a mutation of position 92 in the P chain. This mutation destabilizes the T state, so the conformational state of Hb Chesapeake is biased to the R state, and hence has increased affinity for oxygen. No serious clinical consequences of this mutation have been found apart from lifelong erythrocytosis.
The most common mutations responsible for high-affinity hemoglobins are those that interfere with the R^T transition: mutations in the a2P2 contact area, and in the switch region of the molecule, particularly the C and N terminals, favoring the R state. In addition, mutations that interfere with the 2,3-DPG binding site in the central cavity will tend to increase the affinity of hemoglobin for oxygen.
Hemoglobins with low oxygen affinity (e.g. Hb Kansas, Hb Beth Israel) deliver oxygen more efficiently than normal hemoglobin, hence they tend to be detected as generating hyperoxia. The patient becomes anemic through a correction of the level of erythropoietin secretion. Nevertheless, if the shift to the right is far enough, there is a point at which the delivery of oxygen is normal again and no anemia exists. However, patients with Hb Kansas and Hb Beth Israel have clinically apparent cyanosis, since they have more than 5 g of circulating deoxyhemoglobin. Except for cyanosis, no other abnormalities have been found in these patients. The diag nosis is important in order to avoid unnecessary and sometimes invasive investigations. Diagnosis is by electrophoresis (helpful only if the amino acid substitution affects the overall charge of the molecule) and measurement of the oxygen dissociation curve.
The oxygen delivery properties of high- and low-affinity hemoglobins are discussed in Figure 14.7.
These hemoglobins are the product of the substitutions of either the proximal or distal histidine by tyrosine. Also a nearby mutation, as in Hb M Milwaukee, may produce a similar picture. The patients appear slate grey in color, which may be confused with cyanosis (this is really pseudocyanosis), due to the increase in the deoxyhemoglobin (>5 g/dl) or methemo-globin status of the mutated chains as well as the change of the electrical environment, and hence of the visible absorption spectrum (Figure 14.8). The visible spectra of the hemolysate may help confirm the diagnosis, although electrophoresis can be helpful. Differential diagnosis includes genetic or acquired methemoglobinemia and sulfhemoglobinemia.
These mutations have been found worldwide, and they are rare, except in the Iwate prefecture in Japan where they are common.
Genetically abnormal hemoglobins present themselves to the
Low oxygen affinity hemoglobins
20 40 60
Oxygen tension (mmHg)
Fig. 14.7 Oxygen binding curves of hemoglobins with high and low oxygen affinity
Notice that the extraction of oxygen by the tissues, which is the difference between pulmonary oxygen pressure (100 mmHg) and capillary oxygen pressure (40 mmHg), is lower than normal in high-affinity hemoglobins (low p50) and higher than normal in low-affinity hemoglobins. This is why the former have erythrocytosis and the latter (most of the time) anemia.
Hemoglobinopathies due to structural mutations 171
Hemoglobin M Boston
Hemoglobin M Milwaukee
Heme environment of two Hb Ms compared with Hb A. Notice the diverse changes in the distal and proximal tyrosines (which have replaced the normal histidines) in each of the Hb Ms. These hemoglobins have a characteristic visible spectrum around 610 nm.
F Helix E Helix F Helix E Helix clinician usually in one or more of the following syndromes: (1) the presence of the homozygous or double heterozygous state of Hb S produces a picture of chronic anemia, chronic and insidious organ damage punctuated by painful crises and other complications; (2) the presence of Hb C in the homozygous state produces a mild chronic hemolytic syndrome; when doubly heterozygous with Hb S (SC disease), a syndrome similar to sickle cell anemia but milder, and characterized by microcytic anemia, is seen; (3) the presence of homozygous Hb E produces mild microcytic hemolytic anemia, but in combination with P-thalassemia produces a picture sometimes of very severe thalassemia intermedia; (4) heterozygous forms of unstable hemoglobins produce variable-intensity hemolytic anemias; (5) heterozygotes for low oxygen affinity mutant hemoglobins and Hb Ms may produce a mild hemolytic anemia syndrome, but always with cyanosis or pseudocyanosis as a background; (6) heterozygous forms of high oxygen affinity mutant hemoglobins often present with erythrocytosis.
This panoply of clinical syndromes is the product of the following alterations of the hemoglobin molecule:
• the creation of a new property for the hemoglobin molecule (Hb S, polymerization; Hb C, crystallization and microcytosis)
• changes in O2 affinity (high and low affinity for ligands)
• changes in the environment of the heme (Hb Ms)
• changes in the stability of the molecule in solutions (unstable hemoglobins)
• some mutated hemoglobins are produced at lower rates and generate a thalassemic syndrome (Hb E). Of all of these, only Hb S, Hb C and Hb E are frequent in populations at risk, due to their selection by malaria; the oth -ers are rare and generally represent private mutations.
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