It is now necessary briefly to relate the remarkably diverse molecular pathology described in the previous sections to the phenotypes observed in patients with these diseases. It will not be possible to describe all these complex issues here. Rather we shall focus on those aspects that illustrate the more general principles of how abnormal gene action is reflected in a particular clinical picture. Perhaps the most important question that we will address is why patients with apparently identical genetic lesions have widely differing disorders, a problem that still bedevils the whole field of medical genetics, even in the molecular era.
Beginnings: the molecular pathology of hemoglobin 13
As we have seen, the basic defect that results from the 200 or more different mutations that underlie these conditions is reduced P globin chain production. Synthesis of the a globin chain proceeds normally and hence there is imbalanced globin chain output with an excess of a chains (Figure 1.8). Unpaired a chains precipitate in both red cell precursors and their progeny with the production of inclusion bodies. These interfere with normal red cell maturation and survival in a variety of complex ways. Their attachment to the red cell membrane causes alterations in its structure, and their degradation products, notably heme, hemin (oxidized heme) and iron, result in oxidative damage to the red cell contents and membrane.
These interactions result in intramedullary destruction of red cell precursors and in shortened survival of such cells as they reach the peripheral blood. The end result is an anemia of varying severity. This, in turn, causes tissue hypoxia and the production of relatively large amounts of erythropoietin; this leads to a massive expansion of the ineffective bone marrow, resulting in bone deformity, a hypermetabolic state with wasting and malaise, and bone fragility.
A large proportion of hemoglobin in the blood of P tha-lassemics is of the fetal variety. Normal individuals produce about 1 % of Hb F, unevenly distributed among their red cells. In the bone marrow of P thalassemics, any red cell precursors that synthesize y chains come under strong selection because they combine with a chains to produce fetal hemoglobin and
Selective survival of HbF-containing precursors
Destruction of RBC precursors a i" N
Increased levels of HbF in red cells
High oxygen affinity of red cells
Splenomegaly (pooling, plasma volume expansion)
Skeletal deformity Increased metabolic rate Wasting Gout Folate deficiency
Fig. 1.8 The pathophysiology of P thalassemia
Endocrine deficiencies Cirrhosis Cardiac failure therefore the degree of globin chain imbalance is reduced. Furthermore, the likelihood of y chain production seems to be increased in a highly stimulated erythroid bone marrow. It seems likely that these two factors combine to increase the relative output of hemoglobin F in this disorder. However, it has a higher oxygen affinity than hemoglobin A and hence patients with (3 thalassemia are not able to adapt to low hemoglobin levels as well as those who have adult hemoglobin.
The greatly expanded, ineffective erythron leads to an increased rate of iron absorption; this, combined with iron received by blood transfusion, leads to progressive iron loading of the tissues, with subsequent liver, cardiac and endocrine damage.
The constant bombardment of the spleen with abnormal red cells leads to its hypertrophy. Hence there is progressive splenomegaly with an increased plasma volume and trapping of part of the circulating red cell mass in the spleen. This leads to worsening of the anemia. All these pathophysiological mechanisms, except for iron loading, can be reversed by regular blood transfusion which, in effect, shuts off the ineffective bone marrow and its consequences.
Thus it is possible to relate nearly all the important features of the severe forms of ( thalassemia to the primary defect in globin gene action. But can we also explain their remarkable clinical diversity? Part of it reflects the different mutations of the ( globin genes. For example, some of the promoter or splice mutations cause an extremely mild form of (+ thalassemia. Many ( thalassemics are compound heterozygotes for either two severe ( thalassemia alleles, a severe and mild allele, or different mild alleles, and this also accounts for a considerable amount of clinical diversity of the disease.
What of patients who have the same mutations at their ( loci yet have completely different clinical phenotypes? The co-inheritance of a thalassemia, which reduces the magnitude of the excess of a globin chains in ( thalassemia, may ameliorate the clinical course. This remarkable experiment of nature provides unequivocal confirmation that the major pathophysiological mechanism that underlies ( thalassemia is imbalanced globin chain synthesis. In other patients, especially those who are homozygous for (0 thalassemia yet run a particularly mild course, it is apparent that unusually increased production of fetal hemoglobin is the main ameliorating factor. Although the precise mechanism is not yet understood, it is becoming apparent that genetic determinants both within the ( globin gene cluster and on other chromosomes may be involved in this more effective production of Hb F. For example, a promoter polymorphism of the Gy globin gene may be associated with increased propensity to synthesize fetal hemoglobin, particularly in states of hemopoietic stress. Similarly, there is good evidence that a so-far unidentified gene on chromosome 6 may be involved in modifying the ( thalassemia phenotype in this way. Undoubtedly other polymorphisms of this kind will be discovered.
There is increasing evidence that the complications of P thalassemia may be modified by polymorphisms at different loci. For example, variations at several loci that determine bone metabolism may be involved in modifying the severity of osteoporosis, a common complication of this disorder. Similarly, the occurrence of jaundice and iron loading from the intestine, both common complications of the intermediate forms of P thalassemia, are related to polymorphisms of genes involved in bilirubin and iron metabolism, respectively.
Recently it has been suggested that these complex layers of modifiers of P thalassemia should be placed in three classes: primary, including the different mutations that involve the P globin gene; secondary, including variation at the a globin locus or in fetal hemoglobin production; and tertiary, including modifiers that are involved in varying the severity of the different complications of the disease.
In short, while we have a reasonable idea of how the P thalassemia phenotype is modified, many questions remain and a considerable amount of the clinical variability of the disease remains unexplained.
The pathophysiology of the a thalassemias differs from that of the P thalassemias mainly because of the properties of the excess globin chains that are produced as a result of defective a chain synthesis. While the excess a chains produced in P thalassemia are unstable and precipitate, this is not the case in the a thalassemias, in which excess y chains or P chains are able to form the soluble homotetramers y4 (Hb Bart's) and P4 (Hb H) (Figure 1.9). Although these variants, particularly
High oxygen affinity—hypoxia Instability of homotetramers Inclusion bodies. Membrane damage Shortened red cell survival—hemolysis Splenomegaly—hypersplenism
Fig. 1.9 The pathophysiology of a thalassemia
Hb H, are unstable and precipitate in older red cell populations, they remain soluble sufficiently long for the red cells to mature and develop relatively normally. Hence there is far less ineffective erythropoiesis in the a thalassemias and the main cause of the anemia is hemolysis associated with the precipitation of Hb H in older red cells. In addition, of course, there is a reduction in normal hemoglobin synthesis, which results in hypochromic, microcytic erythrocytes. Another important factor in the pathophysiology of the a thalassemias is the fact that Hb Bart's and Hb H are useless oxygen carriers, having an oxygen dissociation curve similar to that of myoglobin. Hence the circulating hemoglobin level may give a false impression of the oxygen-delivering capacity of the blood and patients may be symptomatic at relatively high hemoglobin levels.
The different clinical phenotypes of the a thalassemias are an elegant example of the effects of gene dosage (Figure 1.10). The heterozygous state for a+ thalassemia is associated with minimal hematological changes. That for a0 thalassemia (the loss of two a globin genes) is characterized by moderate hypochromia and microcytosis, similar to that of the P tha-lassemia trait. It does not matter whether the a genes are lost on the same chromosome or on opposite pairs of homologous chromosomes. Hence the homozygous state for a+ tha-lassemia, - a/-a, has a similar phenotype to the heterozygous state for a0 thalassemia (— /aa).
The loss of three a globin genes, which usually results from the compound heterozygous states for a0 and a+ thalassemia, is associated with a moderately severe anemia with the production of varying levels of hemoglobin H. This condition, hemoglobin H disease, is characterized by varying anemia and splenomegaly with a marked shortening of red cell survival.
Finally, the homozygous state for a0 thalassemia (— /—) is characterized by death in utero or just after birth, with the clinical picture of hydrops fetalis. These babies produce no a chains and their hemoglobin consists mainly of Bart's with variable persistence of embryonic hemoglobin. This is reflected in gross intrauterine hypoxia; although these babies may have hemoglobin values as high as 8-9 g/dl, most of it is unable to release its oxygen. This is reflected in the hydropic changes, a massive outpouring of nucleated red cells, and hepatosplenomegaly with persistent hematopoiesis in the liver and spleen.
While most structural hemoglobin variants produce no clinical disability, a few, notably the sickling, and the rare variants are associated with instability or abnormal oxygen transport (discussed in detail in Chapter 14).
a0 Thal. trait a0 Thal. trait a0 Thal. trait
a0 Thal. trait
Normal a0 Thal. trait a0 Thal. trait
Normal a0 Thal. trait a0 Thal. trait
Hb Bart's hydrops a0 Thal. trait a+ Thal. trait a0 Thal. trait
a+ Thal. trait
Normal a0 Thal. trait a+ Thal. trait
Normal a0 Thal. trait a+ Thal. trait
Hb H disease
Fig. 1.10 The genetics of the common forms of a thalassemia
The light boxes represent normal a genes and the shaded boxes deleted a genes. The mating shown at the top shows how two a0 thalassemia heterozygotes can produce a baby with the hemoglobin Bart's hydrops syndrome. In the mating at the bottom, between individuals with a0 and a+ thalassemia, one in four of the offspring will have hemoglobin H disease.
The sickling disorders represent the homozygous state for the sickle cell gene, sickle cell anemia, and the compound heterozygous state for the sickle cell gene and various structural hemoglobin variants, or P thalassemia. The chronic hemolysis and episodes of vascular occlusion and red cell sequestration that characterize sickle cell anemia can all be related to the replacement of the normal P6 glutamic acid by valine in hemoglobin S. This causes a hydrophobic interaction with another hemoglobin molecule, triggering aggregation into large polymers. It is this change that causes the sickling distortion of the red blood cell and hence a marked decrease in its deformability. The resulting rigidity of the red cells is responsible for the vaso-occlusive changes that lead to many of the most serious aspects of all the sickling disorders.
The different conformations of sickle cells (banana-shaped or resembling a holly leaf) reflect different orientations of bundles of fibres along the long axis of the cell, the three-dimensional structure of which is constituted by a rope-like polymer composed of 14 strands. The rate and extent of polymer formation depend on the degree of oxygenation, the cellular hemoglobin concentration, and the presence or absence of Hb F. The latter inhibits polymerization and hence tends to ameliorate sickling. Polymerization of Hb S causes damage to the red cell membrane, the result of which is an irreversibly sickled cell. Probably the most important mechanism is cellular dehydration resulting from abnormalities of potassium/chloride cotransport and Ca2+-activated potassium efflux. This is sufficient to trigger the Ca2+-dependent (Gardos) potassium channel, providing a mechanism for the loss of potassium and water and leading to cellular dehydration.
The vascular pathology of the sickling disorders is not entirely related to the rigidity of sickled red cells, however. There is now a wealth of evidence that abnormal interactions between sickled cells and the vascular endothelium play a major role in the pathophysiology of the sickling disorders. Recently it has been demonstrated that nitric oxide may also play a role in some of the vascular complications of this disease. It has been found that nitric oxide reacts much more rapidly with free hemoglobin than with hemoglobin in erythrocytes and therefore it is possible that such decompartmentalization of hemoglobin into plasma, as occurs in sickle cell disease and other hemolytic anemias, diverts nitric oxide from its homeo-static vascular function.
These issues are discussed in greater detail in Chapter 14.
There are a variety of different mechanisms underlying hemoglobin stability resulting from amino acid substitutions in different parts of the molecule. The first is typified by amino acid substitutions in the vicinity of the heme pocket, all of which lead to a decrease in the stability of the binding of heme to globin. A second group of unstable variants results from amino acids that simply disrupt the secondary structure of the globin chains. About 75% of globin is in the form of a helix, in which proline cannot participate except as part of one of the initial three residues. At least 11 unstable hemoglobin variants have been described that result from the substitution of proline for leucine, five that are caused by an alanine-to-proline change, and three in which proline is substituted for histidine. Another group of variants that causes disruption of the normal configuration of the hemoglobin molecule involves internal substitutions that somehow interfere with its stabilization by hydrophobic interactions. Finally, there are two groups of unstable hemoglobins that result from gross structural abnormalities of the globin subunits; many are due to deletions involving regions at or near interhelical corners. A few of the elongated globin chain variants are also unstable.
There is a family of hemoglobin variants that are associated with high oxygen affinity and hereditary polycythemia. Most result from amino acid substitutions that affect the equilibrium between the R and T states (see earlier section—Structure and function). Thus, many of them result from amino acid substitutions at the at/P2 interface, the C terminal end of the P chain, and at the 2,3-DPG binding sites.
There is a family of structural hemoglobin variants that is designated hemoglobin M, to indicate congenital methemo-globinemia, and is further defined by their place of discovery. The iron atom of heme is normally linked to the imidazole group of the proximal histidine residue of the a and P chains. There is another histidine residue on the opposite side, near the sixth coordination position of the heme iron; this, the so-called distal histidine residue, is the normal site of binding of oxygen. Several M hemoglobins result from the substitution of a tyrosine for either the proximal or distal histidine residue in the a or P chain.
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