- Email: [email protected]
Chapter 2 Hemolytic anemias due to hemoglobinopathies
Chapter
2
Hemolytic Anemias Due to Hemoglobinopathies Claude Poyart* and Henri Wajcmant *INSERM U299, Hdpital de Bicktre, 94275 Le Kremlin Bic&re, France, tlNSERM U91, HGpital Henri Mondor, 94070 Crbteil, France
Abstract-Hemoglobinopathies responsible for hemolytic anemias may be divided into two groups. The first one corresponds to thalassemias and the second to the presence of a structurally abnormal hemoglobin (Hb). In thalassemia, the primary biochemical abnormality is a quantitative defect in the biosynthesis of one type of Hb chain. This defect leads to an overall deficit of Hb accumulation in the erythrocyte (hypochromia) together with the presence of an excess of the normally synthesized chains. The unpaired subunits which are less soluble than HbA precipitate, bind to the membrane and ultimately lead to hemolysis. In the second group, the hemolytic anemia is a direct consequence of the physicochemical properties of the structurally abnormal Hb. This molecule may polymerize, precipitate or crystallize within the red blood cell (RBC) leading to membrane alterations and to the destruction of the cell. This chapter will emphasize several examples of structurally abnormal Hbs, such as sickle cell disease and congenital Heinz body hemolytic anemia (CHBHA).
Molecular Genetics Molecular
genetics of thalassemias
Thalassemias are the most common of all the Hb disorders (Weatherall and Clegg, 1981). They are primarily found in populations originating from Mediterranean, African or Asian countries. The high prevalence of the various thalassemic genes in these populations is probably explained by a selective pressure exerted by malaria. Thalassemias are classified according to the type of chain which is affected: the two main categories consist of LX-and /?-thalassemias: each can be subdivided into several different subtypes.
Genetics of or-thalassemia The genetics genes (Higgs
of cc-thalassemia are most frequently explained by the deletion of a et al., 1989). Normal individuals have two CI genes (W/W) on each 129
130
Claude Poyart and Henri Wajcman
chromosome 16, which are named ct2 and xl, from 5’ to 3’. The a2 gene encodes two- to three-fold more protein than the xl gene. Carriers of cc-thalassemia have either three (-~/GYM)or two (--/w or -Z/-(X) r genes. These individuals having only two functional genes are now classified as being heterozygous for an a”-thalassemia or homozygous for an r+ -thalassemia, respectively. The a-thalassemic patients suffering from hemolytic anemia usually have an excess of free p chain tetramers (HbH) in their red blood cells (RBC) because of the deletion of three 9 genes (-l-x). The homozygous situation for a”-thalassemia leads to Bart’s hydrops fetalis syndrome responsible for fetal death ir2 utero. A consequence of these genetic exchanges is the existence of individuals clinically normal and having five or six genes. Deletions within the x gene cluster may be favored by the presence of many repetitive sequences, with a surprisingly high G+C content (Lauer et al., 1980). They may result either from reciprocal recombination between two chromosomes or from gene conversion involving the two genes of the same cluster. The region involving the two c( genes may be divided into homologous subsegments (X, Y and 2) and nonhomologous elements (I, II and III). Recombination between 2 segments results in cr+-thalassemic chromosomes having the ~~~~~ rightward deletion (Fig. 4). Recombination between the homologous X segment gives rise to the -a4.’ leftward deletion. In both cases, several subtypes may be distinguished according to the exact their point of recombination. Homozygotes for the - c(‘.’ deletion have theoretically two a2 genes missing but the amount of x-globin synthesized is higher than the 25% keep expected. Conversely, homozygotes for the - r3.7 deletion, which theoretically of the equivalent of two ct2 genes, express only about half of the normal quantity which involve cc-globin instead of the expected 75% (Bowden et al., 1987). Deletions
w
a2
a2
al
al enli-a 3.7
-a 3.7 ylal
a2
al
between the two a Fig. 4. A mispairing in the homologous regions followed by recombination genes leads to a deletional a-thalassemia. In this example the mechanism involving the 2 homology blocks that gives the -a “’ rightward deletion is shown. X, Y, Z and I, II, III are the homologous and non-homologous regions, respectively. The distance between the two X blocks and the two 2 blocks is 4.2 and 3.7 Kb, respectively.
Hemolytic Table 1. Some unstable
*: chain variants responsible
Variant
Structural
Hb Hb Hb Hb Hb Hb Hb Hb
r14(A12) Trp+Arg r29(BlO) Leu+Pro 238 or 39(C4) Thr+0 a59(Gll) Gly-+Asp a104(E8) Cys+Tyr a109(G16) Leu+Arg ~llO(G17) Leu+Asp x125(H8) Leu+Pro
Evanston Agrinio Taybe Adana Sallanches Suan Dok Petah Tikva Quong Sze
*Compound
heterozygote
131
Anemias Due to Hemoglobinopathies
modification
for r-tha12, **homozygote,
for non-deletional
% HbH
Gene Rightward x2 Xl xl a2 r2 ? x2 ***compound
thalassemias
r-tha12
heterozygote
Traces Traces* 0.5** Traces*** 2** 15*** 3*** 35*** for a-thall.
both the x.2 and the ctl genes are responsible for x0-thalassemia. Several types been described that may be distinguished by their length and starting point.
have
Many examples of a-thalassemic patients with HbH disease have been found in which one of the CI genes is present but not functional. The reason is the presence of a mutation(s) in a control region affecting the expression of this gene. Several examples are known of such non-deletional a-thalassemic genes. As shown in Table 1, the same picture may be produced by the presence of a mutation encoding for an unstable protein (Kan et a/., 1977).
Genetics of /I-thalassemia In the b-thalassemias the gene is almost always present. A great variety of mutations have been described that may affect any of the regions involved in the control of the expression of the gene (Huisman, 1992). When the expression of the gene is totally abolished, the defect is known as PO-thalassemia and when globin is still synthesized in small amounts as /3+-thalassemia. HbE is both a structurally abnormal hemoglobin and a /I+-thalassemia (Orkin, 1987). In this example the codon 26 located at the end of the first exon is both a missense (Glu-+Lys) and an alternative site for splicing. Usually no hemolytic anemia is observed in the heterozygous carriers of a fl-thalassemic gene, the red cell parameters showing only a mild hypochromia and microcytosis. In contrast, a severe hemolytic anemia with a high degree of bone marrow stimulation is observed in patients that are either homozygotes or compound heterozygotes for the two different /I-thalassemic genes. The severity of the hemolytic process may vary to a large extent from the classical Cooley anemia that requires blood transfusion and iron chelation for life, to that of thalassemia intermedia in which blood transfusion is usually not necessary. In patients that associate both fi-thalassemic and x-thalassemic genes, the biosynthesis of both kinds of chains is decreased and, as a result, a lower amount of unpaired subunits accumulates in the RBC which are still hypochromic but the hemolytic process is less severe. Conversely when a patient associates a p-thalassemia and a triplication of the r genes in one of his chromosome 16 the clinical picture may be a thalassemia intermedia.
132
Claude Poyart and Her-vi Wajcman
The genetic mechanism for G/?-thalassemias is a deletion involving totally or partially both 6 and p genes. In these patients the expression of the y genes is usually high and the small amount of adult Hb is partly compensated by an increased synthesis of fetal Hb. As a result the hemolytic anemia syndrome is mild as in /Y-thalassemias.
Molecular genetics of abnormal hemoglobins From a genetic point of view, abnormal Hbs and thalassemia inherited. From a clinical point of view this is less evident carriers of thalassemic genes and of several abnormal Hbs are the biological consequences of the abnormahty may be difficult
are both codominantly since the heterozygous healthy. In some cases to identify.
A4olecular genetics of sickle cell disease HbS is the most frequent abnormal hemoglobin found in populations of African origin but is also observed in Arab and Indian populations. HbS results from the substitution of a valine for a glutamic acid at position 6 of the fl chain. HbS has a multi-centric origin (Pagnier et al., 1984) and appeared independently in at least five locations. Heterozygous carriers for HbS are usually healthy and detected only by biological and clinical hemoglobin studies. Clinical manifestations of the abnormal hemoglobin are exceptional, except for kidney lesions characterized by interstitial papillary necrosis (Lantz et al., 1995). Conversely, patients suffering from homozygote sickle cell disease exhibit a severe hemolytic anemia and varying secondary complications affecting all the organs by thrombo-embolism. This is also the situation for compound heterozygotes for HbS and a J?-thalassemic gene, or for individuals carrying in addition to HbS an additional mutation that enhances the polymerization processes of sickle cell Hb (Serjeant, 1985). The presence of various genetic factors that stimulate the production of fetal Hb (HPFH-like genes) leads to a sickle cell disease that is better tolerated than others. Five factors have been shown to influence the high variation of HbF levels in sickle cell anemia: age, sex, the IX globin gene number, ,O globin haplotypes and mostly an X-linked factor that regulates the production of HbF-containing erythrocytes (Chang et al., 1995). Since HbS is a frequent abnormality, several examples have been identified in which another genetic event, such as a second globin point mutation or a crossover with a gene carrying another gene, occurs in the same gene. This situation has been well documented in the case of HbC Harlem and HbS Antilles. Both variants carry the substitution of HbS. In the first case, it is associated with the mutation of Hb Korle Bu which possesses an anti-sickling effect (Bookchin et al., 1967). In the second case, the /36 Glu-+Val mutation is associated with the substitution of an Ile for a Val at position p23 (BS), which enhances HbS polymerization (Montplaisir et al., 1986). Many Hb variants, like HbS, are well tolerated in the heterozygous form but induce in homozygous carriers a mild hemolytic anemia, such as encountered HbC, which cnjstallizes within the red blood cells.
may in
Hemolytic Anemias Due to Hemoglobinopathies
133
Molecular genetics of hemolytic anemia due to unstable hemoglobins By contrast, unstable abnormal Hbs behave frequently as hereditary dominant. These Hbs have a structurally abnormal chain resulting in a molecular instability which causes CHBHA (Bunn and Forget, 1986). The heterozygous carriers suffer from a hemolytic anemia, the severity of which depends on the structural abnormality induced by the mutation (Ohba, 1990). In some cases, the anemia is very mild but in other cases is life threatening requiring frequent blood transfusions. According to the International Hemoglobin Information Center (199.5), approximately 200 different mutations have been identified as responsible for unstable Hbs.
Unstable Hbs result from private mutations limited to a few individuals or to a small number of families. Therefore, de nouo cases are not exceptional. Abnormal Hbs with identical pri.mary sequence structural abnormalities, as for example in Hb Koln [/I 98 (FG5) Val+Met], have been found in several unrelated patients. This is likely to be a consequence of the presence of hotspots for mutations. The frequency of mutations involving CpG dinucleotides seems to be higher than random (Perutz, 1990). The high number of de nouo cases of unstable well-characterized Hbs variants may, however, be biased relative to other Hb variants, by the fact that the Hb status of patients suffering from a hemolytic anemia is thoroughly investigated. The availability of sophisticated methods helps considerably to identify mutations that may otherwise behave as neutral with routine electrophoretic tests. In other cases, several siblings from apparently normal parents are affected; this may be explained by a germline mosaicism due to a somatic mutation having occurred at an early stage of the embryologic development of one of the parents (Wajcman et al., in press). Unstable Hbs may also result from several mechanisms, such as the simultaneous presence of two mutations in the same gene or from the addition or deletion of short sequences. These more complex genetic events may be explained by the presence of DNA sequences having a sufficient degree of homology to allow mispairing followed by breakage and incomplete reparation (Cooper and Krawczak, 1991; Krawczak and Cooper, 1991).
Unstable hemoglobins and dominant thalassemias When the instability of the CX-or /I-globin chains is very high, with a half-life of only a few minutes or hours, the molecule is denatured and lost in the red cell precursors of the bone marrow and the clinical expression becomes similar to that of a thalassemia intermedia. The main difference with a thalassemic syndrome consists of the genetic inheritance which follows a clinically dominant mode. These Hbs are indeed often classified as dominant thalassemias. In the cases where the half-life is slightly longer, all the abnormal Hb may be eliminated in the spleen and all the tests for an unstable Hb can be negative in the peripheral blood. In some of these patients, the abnormal Hb has been detected only after splenectomy.
134
Claude Poyart and Henri Wajcman
Pathophysiological Mechanisms The pathophysiology of hemolysis shares a common mechanism in the various hemoglobinopathies. In unstable Hbs, the structurally abnormal Hbs form precipitates that are bound by the membrane skeleton, decreasing the cell deformability and revealing abnormal epitopes favoring lysis. In the case of thalassemia, the free subunits, which are in excess, behave like an unstable Hb. In sickle cell disease, the polymerized molecules of deoxy HbS alter the shape of the cell and the structure of the membrane, leading to membrane loss and ultimately to cell destruction. In all these cases, the hemolysis is intra- and mostly extravascular. The patients suffer from a chronic hemolysis. In normal conditions and due to efficient bone marrow compensation, the anemia is well tolerated.
Genera/ mechanisms
for extravascular hemolysis
Heinz body formation Heinz bodies are the products of Hb degradation. The precipitated material is made of hemichromes, which are low spin derivatives of ferric Hb (Rachmilewitz, 1974). In some of them, the sixth coordination position of the heme is occupied by a ligand that may be a hydroxyl, a water molecule, a protonated or an unprotonated histidyl provided by the globin. Hemichromes are generated when the heme dissociates from its normal position in the heme pocket and rebinds elsewhere in the globin. Denatured hemoglobin binds with extremely high affinity to proteins of the erythrocyte membrane, principally to the cytoplasmic portion of band 3 (also named erythrocyte anion exchange channel) (Waugh and Low, 1985). This causes band 3 to aggregate, forming clusters that are responsible for the appearance of senescence antigens on the surface of the cell. These epitopes are recognized by macrophages. Deposits of immunoglobulins specific to these abnormal epitopes can be responsible for some degree of immunological lysis of the cell in conjunction with complement. Heinz bodies also generate many oxidant agents (superoxide, peroxide or hydroxyl radicals) that damage the proteins and the lipids of the membranes. The presence of Heinz bodies decreases the deformability of the erythrocyte and enhances its fragility; two factors leading to the destruction of the cell in the spleen that will be considered below.
Spleen and hemolysis The extravascular hemolytic process is mediated by the macrophages of the bone marrow, the liver and the spleen. Severe unstable Hbs, in which the subunits are not able to assemble into soluble tetramers, result in a high degree of hemolysis in the bone marrow. In contrast when the tetramers become unstable following oxidative stress, the destruction of the RBCs occurs mainly in the spleen. Normally, all the circulating RBCs are controlled by the spleen allowing the removal of aged or damaged cells (Chen and Weiss, 1973). Only the erythrocytes, which have a normal deformability, may pass through the filter of the spleen without damage. Poorly deformable cells or cells containing precipitated materials are not able to pass
Hemolytic
Anemias Due to Hemoglobinopathies
135
safely between cordal macrophages that recognize abnormal membrane epitopes. These cells also have difficulties passing through the fenestrations of the basement membrane of the sinus and between the endothelial cells. The spleen, in addition to its culling function, e.g. the removal of abnormal and deleterious cells, has a pitting function that consists of removing inclusion bodies formed within the cells. When a erythrocyte containing inclusion bodies attempts to pass through the sinusoidal filter, the inclusions accumulate in the last part of the cell which can no longer enter the sinus. This part of the cell which has lost its deformability is then removed by fragmentation. The fragments released will be digested by the macrophages while the remainder of the RBC returns in the circulating blood. The damaged cells having lost some membrane material are more fragile than normal and will soon become a target for destruction by macrophages. They may also be culled by the spleen in a subsequent passage.
Hemolysis in unstable hemoglobins Substitutions in the primary sequence of the Hb chains can result in a subunit that is unstable and tends to precipitate. These mutations usually affect a functional region critical for the stability of the molecule: examples are the heme pocket, the intersubunit contact areas in the dimers or tetramers or the interior of the globin. Instability may also result from a large structural perturbation due to the disruption * of a helical structure or to an amino acid deletion (Fermi and Perutz, 1981). In the heme pocket different types of structural abnormalities have been described. When the structural abnormality involves the proximal side of the heme, the strength of the bond between the heme and the F helix of the globin is decreased. This is illustrated by several variants, such as Hb Boras [fl88(F4) Leu-+Arg] (Hollender et al., 1969), Hb Bristol [/?67(Ell) Val+Asp] (Steadman et al., 1970) or Hb Olmsted [p141(H19) Leu-*Arg] (Lorkin et al., 1975). Some examples are also known in which the proximal histidine (F8) is replaced by a residue that cannot bind the heme. This leads to the formation of unstable tetramers, named semi-hemoglobins, in which only the normal chains carry a functional heme group. Typical examples of this pathology are Hb Redondo [p92 (F8)His+Asn] (Wajcman et al., 1991) or Hb Saint Etienne [fl92 (F8) His-tGln] (Beuzard et al., 1972). In Hb Redondo, the instability is enhanced by the deamidation of the asparagine residue replacing the F8 histidine, which may lead to some cleavage of the protein backbone. Conversely, when the structural modification is localized on the distal side of the heme, the aperture of the heme pocket may be increased, favoring entry of water which increases the rate of oxidation. Classical examples are provided by Hb Zurich [p63 (E7)His+Arg] (Muller and Kingma, 1961) and Hb Bic&tre [p63 (E7)His-+Pro] (Wajcman et al., 1976) in which the distal histidine is replaced by an arginine and a proline, respectively. In Hb Bic&tre, the rate for autooxidation of the abnormal chains is increased and the tetramers behave as p valency hybrids in which the a chains bind oxygen but the p chains are in the metHb form. Hb Zurich has both an increased autooxidation rate and a much higher than normal affinity for carbon monoxide. Carriers of Hb Zurich that are smokers therefore display a high level of HbCO which
136
Claude Poyart and Henri Wajcman
is much more stable than the metHb. The CHBHA of these patients is therefore very atypical: Heinz bodies in the red cells are rare and the Hb level is normal or even increased due to a high oxygen affinity. Another group of mutations involves residues that participate in subunit interactions. In the tetrameric structure of Hb, the interface between the c( and p subunit (crij3i contact) of each dimer is relatively rigid. Mutations occurring in this region lead to instability because the tetrameric assembly dissociates into dimers and then monomers. These free subunits uncoil, have a high autooxidation rate (16 times that of native HbA) and finally form hemichromes that precipitate. Examples are provided by many variants among which Hb Philly l-835 (Cl)Tyr+Phe] (Rieder et al., 1969) in the p chains or Hb Taybe (Galacteros et al., 1994) in the c1 chains. In the case of a chain variants the unstable Hb may be responsible for the presence, in addition to traces of HbH (but to a much lower extent than in cc-thalassemia) of an artifactual fi-thalassemic-like biosynthetic ratio. The hypothesis of a disturbed association between subunits may conciliate these paradoxical observations. The introduction of a proline, or sometimes of a glycine, in the middle of a helix disrupts the secondary structure and leads to the instability of the Hb molecule. This mechanism is one of the most frequently observed. Mutations that modify the folding interactions between the different CI helical regions or which permit the introduction of additional water molecules which are not present normally inside the Hb, may also modify the solubility of the molecule and lead to instability and the subsequent oxidation and precipitation. Some Hbs are designated as hyperunstable because they are destroyed so rapidly that they are barely detectable or undetectable in the hemolysate. These Hbs usually result from mutations localized in the third exon, in regions coding for the ccl/?1 contact. As shown in Table 2, they include point mutations, insertions or deletions of residues or frameshift. In the case of the double mutant Hb Medecine Lake [P32(B14) Leu-+Gln; /398(FG5) Val-+Met] (Coleman et al., 1995) the same globin chain carries two substitutions that even when present alone are responsible for CHBHA. Some of these hyperunstable variants may be detected after splenectomy in the hemolysate.
Hemolysis in thalassemia The reduced synthesis of one of the globin chains results in an overall deficit in Hb accumulation and to the presence of a large excess of the non-affected globin chain. These free subunits form unstable aggregates that precipitate within the cell leading to the above described membrane disorders.
P-thalassemia In P-thalassemia, the inclusions bodies contain aggregated a-chains. They are formed of the bone marrow, leading to ineffective in the etythrocyte precursors erythropoiesis. Usually, these Heinz bodies have disappeared from the cytosol of the activity is high in the erythrocyte circulating erythrocytes since the protease By contrast, they are often observed in bone marrow smears. In this precursors.
137
Hemolytic Anemias Due to Hemoglobinopathies regard, thalassemic syndromes differ from unstable formation occurs most frequently in the peripheral oxidative stress.
Hbs blood
in which Heinz when submitted
body to an
cl-thalassemia In ol-thalassemias, the excess of P-chains form tetramers (Hb H) which are more stable than the free LXchains. As a consequence, ineffective erythropoiesis and bone marrow destruction of the erythrocyte precursor is less marked. The precipitation of Hb H proceeds more gradually and occurs in the peripheral blood rather than in the precursors of the bone marrow. The pathophysiological mechanism of hemolysis, in which the spleen plays a crucial function, becomes, in this case, very similar to that of unstable Hbs.
Hemolysis in sickle cell disease Hb S polymerizes when the cell is submitted to reduced oxygen tension, forming long fibers that modify the shape of the red cells. As long as the membrane of the cell has
Table 2. Mechanism
Substitution involving one residue Chesterfield Cagliari Terre Haute Showa Yakushiji Durham NC Houston Presence of two substitutions Medicine Lake
for hyperunstable
variants affecting the /I’chain
fl28 Leu+Arg /3 60 Val+Glu j 106 Leu-rArg /I 110 Leu-+Pro b 114 Leu+Pro /I127 Ala-Pro
in the same chain /I 32 Leu-+Gln,
fl98 Val+Met
Deletion or addition of one or several codons Korea /3 33 or 34 (Val+O) Gunma codons 127-128 (Gin-Ala+Pro) 0 fi 134-137 (- 12, +6) (Val-Ala-Gly-Val-Gly-Arg) Koriyama /? 95(+Leu-His-Cys-Asp-Lys-) 96 Early chain termination 0 0 Frameshift by deletion Agnana 0 Manhattan Geneva Makabe Kohn Kaen Vercelli 0
codon 121 (GAA-+TAA: codon 127 (CAG+TAG:
Glu-+Term) Gln-rTerm)
or addition of one or several nucleotides codon 94 (+TG) + 156 residues long chain codons 106-107 (+G) codon 109 (-G) -+ 156 residues long chain codon 114 (-CT, +G) + 156 residues long chain codon 123 (-A) + 156 residues long chain codons 123-125 (-ACCCCACC)+156 residues long chain codon 126 (-T) +156 residues long chain codons 128 (-4,+5, -11) + 153 residues long chain
138
Claude Poyart and Henri Wajcman
not been too severely injured, sickling is reversible upon oxygenation. After several oxy-deoxy cycles, the membrane alterations accumulate and sickling becomes an irreversible process. The irreversibly sickled cells are dehydrated dense cells containing a large amount of polymerized Hb S. Their number correlates well with the hemolytic rate and the spleen size. In normal red cells the phospholipids of the membrane are asymmetrically distributed: amino phospholipids (phosphatidyl serine and phosphatidyl ethanolamine) the inner layer and choline-phospholipids are mainly in (phosphatidylcholine and sphingomyelin) in the external one. As a consequence of the injuries produced by the Hb S polymers, this asymmetric composition is barely maintained. This abnormal lipid partitioning leads to several consequences among which are an enhancement of the procoagulant activity and an increased adhesion to the macrophages. Hb S also leads to the formation of micro-Heinz bodies, which are much smaller than those observed in unstable Hbs. These inclusion bodies generate potent oxidants which alter the structure of the membrane. In addition, they bind with a high affinity to the cytoplasmic region of band 3 causing it to aggregate. The distribution and nature of the surface epitopes of the RBC is therefore modified. This process has been involved in the formation of senescence antigens leading to hemolysis.
Clinical Picture An extensive literature is already available on the clinical presentation of thalassemias and sickle cell disease and it is out of the scope of this short review to develop these points. Therefore, we will limit this chapter to the clinical picture of unstable Hbs.
Historical The first case of congenital hemolytic anemia due to an unstable Hb has been reported by Cathie (1952). It concerned a baby that was 10 months old when diagnosed. The child presented jaundice, splenomegaly and emission of dark urines. This observation differed from familial spherocytosis because the hematological disorders did not disappear after splenectomy. The biochemical identification of the molecular abnormality was discovered only 18 years later when it was shown that the baby carried an abnormal Hb (named Hb Bristol), in which the valine residue at position /?67 (El 1) was replaced by an aspartic acid (Steadman et al., 1970).
Biological
diagnosis of an unstable Hb
The discovery of Heinz bodies in blood smears after supra Hb. The presence suggest the presence of an unstable nevertheless not pathognomonic of an unstable Hb and several red cell enzymopathies, are also known to produce them.
vital staining usually of Heinz bodies is mechanisms, such as
The definition of an unstable Hb is a biochemical one: it means that the solubility of the Hb molecule is reduced as compared to that of Hb A (Huisman and Jonxis, 1977).
Hemolytic Anemias Due to Hemoglobinopathies
139
Thus, historically, an unstable Hb is a molecule that precipitates when incubated for 1 hour at 50°C. In these conditions only the severe unstable Hb are detected. More sensitive tests are now available which lead to positive results for less unstable molecules. One of the most widely used consists of incubating the lysate at 37°C in the presence of 17% isopropanol during a length of time insufficient to precipitate Hb A (Carrel and Kay, 1972). Another accurate method is to record the rate of denaturation of the Hb molecule under standardized conditions of heme concentration and at a higher temperature (65°C). The test should be done on a hemoglobin solution previously equilibrated under 1 atm oxygen for 20-30 min in order to test fully liganded tetramers. When a Hb is very unstable, its amount in the circulating blood may be too low to lead to a clear precipitate and the instability tests may be erroneously considered as negative. The same situation may occur for an unstable Hb that is encoded by a gene expressed at a low level such as the xl gene (Galacteros et al., 1994). Unstable Hbs that have a half-life of only a few hours may be detected by chain biosynthesis measurements. When the reticulocytes of these patients are incubated during a short time period, 1 hour or less, in the presence of a labeled amino acid, the abnormal chain which is synthesized at a normal rate may be demonstrated.
Clinical severity of unstable hemoglobins The clinical disorders and, therefore, several
are directly proportional to the instability of the Hb molecule degrees of severity may be recognized, as shown in Table 3.
In many cases the slightly unstable Hb molecule variants are detected by in vitro studies; these variants are without clinical consequences unless they are associated with another red cell or membrane abnormality or submitted to a strong oxidative stress. In the case of moderately unstable Hbs, the anemia is the most frequently well compensated. The patient may nevertheless suffer from hemolytic crises during infectious episodes likely to be related to pyrexia or transient acidosis. In some cases, according to the structural abnormality involved, an oxidative drug may be the trigger mechanism of a hemolytic crisis. Emission of dark urines is characteristic but not
Table 3. Chnical manifestations
PI PII [III1 F
IYI
of unstable
Hbs (from Ohba, 1990)
Life long severe hemolytic anemia Life long mild to moderate hemolytic anemia Mild hemolytic anemia with slight jaundice Positive test for instability only found during in vitro studies “Dominantly inherited /?-thalassemia“ Recessively inherited r-gene disorders with Hb H traces
140
Claude Poyart and Henri Wajcman
always noticed. In the steady state, the patient does not require except for supportive and preventive measures (administration prevention of infection, avoidance of oxidative drugs).
specific therapy of folic acid,
Frequently the decreased stability is not the only abnormal feature of an unstable Hb molecule. The structural modification may affect a region which is not only crucial for stability but also for oxygen binding. Modified oxygen binding properties are therefore frequently associated with instability. When the affinity for oxygen is increased the oxygen delivery to the tissues is impaired. This leads to a stimulation of erythropoiesis which may increase the Hb level to values close to normal, such a patient will only have an apparently well compensated anemia. In contrast, unstable Hbs with decreased oxygen affinity may appear to be more severe. In the steady state these patients have a significantly lower Hb level than the previous ones and this value may drastically drop during hemolytic crises. Severe hemolytic disorders may require splenectomy and the patients then become submitted to specific complications among which are serious thrombo-embolic which are a major cause of episodes (Beutler et al., 1974). These complications, morbidity in sickle cell disease, have also been reported in thalassemia intermedia and other hemoglobinopathies. Many abnormalities of the RBC, that are present in carriers of unstable hemoglobins, have been implicated as causes of an increased adhesiveness. Membrane sialic acid abnormalities, oxidative membrane damage, loss of membrane phospholipid asymmetry, and binding to various plasma adhesive proteins (fibrinogen, fibronectin, von Willebrand factor) are among these factors. Owing to the exteriorization of procoagulant phospholipids in the RBC membranes, thrombin generation and fibrin formation appear also to be increased whereas antithrombin III, protein C and free protein S are reduced (Francis and Johnson, 1991). In contrast, other variants lead to very severe hemolytic disorders with a red cell life limited to a few days or even to a few hours. These hyperunstable Hb variants are destroyed in the erythroblasts of the bone marrow and result in a syndrome having clinical manifestations similar to those observed in thalassemia intermedia (Adams and Coleman, 1990; Thein et al., 1990; Thein, 1992). All the intermediary forms between these two extremes have been described. Unstable variants due to a structural modification carried by the a-chain have biological and clinical expression varying considerably from one case to another. This may be explained by the fact that or-chains are encoded by two genes. Since the a2 gene encodes for a higher level of mRNA than the cxl gene, a greater impact of an a2 locus mutation is predicted, in comparison to mutations of the al gene. In addition, according to the mechanism of instability involved, an abnormality of the ~2 locus may lead either to a ‘classical unstable Hb syndrome’ or to an cc-thalassemia like phenotype when the Hb is destroyed in the bone marrow. In some ‘thalassemic (Goossens et at., 1981), the variants’, such as Hb Quong Sze [a125 (H8) Leu +Pro] mutated chain is destroyed before assembling with the partner subunit, leading to a pure a-tha12 including some degree of ineffective erythropoiesis (Table 1). In other variants, the clinical picture results from a mixture of two features: (1) an increased erythropoiesis and (2) an early precipitation of the abnormal hemoglobin during RBC
Hemolytic Anemias Due to Hemoglobinopathies
141
maturation. In some cases, such as Hb Questembert [a131 (H14) Ser+Pro] (Wajcman et al., 1993) or Hb Ann Arbor [a80 (Fl) Leu+Arg] (Adams et al., 1972) the structural abnormality may result in a disturbed protein folding and subunit assembly and in paradoxical biosynthetic ratio.
Biological Bases for Therapy In spite of the profound understanding of the molecular pathogenesis of hemoglobinopathies, no efficient specific treatments are available. Nevertheless, recent advances in transfusional programs, in pharmacology, in allogeneic bone marrow transplantation and in molecular genetics offer considerable promises in preventing or delaying most of the complications associated with hemoglobinopathies (Rodgers et al., 1994).
Progress in iron chelation therapy Striking improvement in life expectancy has been obtained in the treatment of thalassemia by hypertransfusion programs when associated with an effective prevention from the toxic effects of iron overload. Up to now the only therapeutically available iron chelating agent is deferoxamine. It needs to be administrated by parenteral mode and the prognosis of a patient submitted to nightly subcutaneous deferoxamine perfusion has been considerably improved. Also, very efficient programs, using pumps for continuous ambulatory intravenous perfusion over several days per month have been designed. An orally active chelator, L- 1 (1,2-dimethyl-3-hydroxypyrid-4-one) has been recently proposed for patients unable to comply with parenteral therapy. A large controlled multicentric study has recently been initiated to determine the efficiency and the toxicity of this drug (Olivieri et al., 1995).
Allogeneic
bone marrow transplantation
Since the initial report in 1982 of a bone marrow transplantation in a thalassemic patient, several hundreds of thalassemic patients, all over the world, have been cured by this treatment. Survival rates have clearly improved with more effective prevention of infections, of graft failure and of graft versus-host-disease. The experience is much more limited with sickle cell disease. The risk of complication of a bone marrow transplant seems now to be sufficiently low to consider such a treatment for prevention of long term morbidity in some selected patients suffering from serious forms of hemoglobinopathies. The inclusion criteria for sickle cell patients to benefit from this therapy have been recently determined (Bernaudin et al., 1995).
Modulation
of globin gene expression
Since Hb F interferes with the polymerization of deoxyHb S in sickle cell disease and compensates for the unbalanced chain biosynthesis of /&thalassemia, several agents
142
Claude Poyart and Henri Wajcman
have been evaluated for their effect in the augmentation of synthesis of Hb F in these hemoglobinopathies. In sickle cell disease, a significant effect of Hb F requires both a total Hb F level of at least 20% with a distribution over 75% of the RBC. To achieve this goal several agents such as cytotoxic agents (5azacytidine or hydroxyurea), hematopoietic growth factors (cytokines, erythropoietin), or short chain fatty acids (arginine butyrate) have been proposed. Hydroxyurea is the least toxic of the cytotoxic agents. It has been used widely for patients with sickle cell disease and it has been demonstrated that most patients respond to this drug by doubling their baseline level of Hb F and F-reticulocyte count (Charache et al., 1992). Long-term therapy seems to decrease the frequency and severity of pain crisis and to improve quality of life of the patients. Results concerning /3-thalassemic patients are less clear. Among the hematopoietic growth factors only recombinant human erythropoietin has been reported in clinical trials (Rachmilewitz et al., 1991). It was demonstrated to increase the percentage of F-reticulocytes and Hb F level in sickle cell patients when administered at high doses along with supplemental iron. Also, erythropoietin exerts an additive effect when administered with hydroxyurea. Clinical trials are underway. Attention has recently been focused on butyrate analogs and other short chain fatty acids. These molecules have been found to induce hematopoietic cell differentiation and to increase Hb F level. Clinical trials with phenylbutyrate alone or in combination with other agents are underway in sickle cell and in thalassemic patients (Perrine et al., 1993).
Genetic treatment Hemoglobinopathies are good candidate diseases for gene therapy since they could be cured by inserting an efficient fl-globin gene in the progenitor cells (Sadelain, 1995). This strategy awaits the solution of several biological problems which are out of the scope of this review.
2
Hemolytic Anemias Due to Hemoglobinopathies Claude Poyart* and Henri Wajcmant *INSERM U299, Hdpital de Bicktre, 94275 Le Kremlin Bic&re, France, tlNSERM U91, HGpital Henri Mondor, 94070 Crbteil, France
Abstract-Hemoglobinopathies responsible for hemolytic anemias may be divided into two groups. The first one corresponds to thalassemias and the second to the presence of a structurally abnormal hemoglobin (Hb). In thalassemia, the primary biochemical abnormality is a quantitative defect in the biosynthesis of one type of Hb chain. This defect leads to an overall deficit of Hb accumulation in the erythrocyte (hypochromia) together with the presence of an excess of the normally synthesized chains. The unpaired subunits which are less soluble than HbA precipitate, bind to the membrane and ultimately lead to hemolysis. In the second group, the hemolytic anemia is a direct consequence of the physicochemical properties of the structurally abnormal Hb. This molecule may polymerize, precipitate or crystallize within the red blood cell (RBC) leading to membrane alterations and to the destruction of the cell. This chapter will emphasize several examples of structurally abnormal Hbs, such as sickle cell disease and congenital Heinz body hemolytic anemia (CHBHA).
Molecular Genetics Molecular
genetics of thalassemias
Thalassemias are the most common of all the Hb disorders (Weatherall and Clegg, 1981). They are primarily found in populations originating from Mediterranean, African or Asian countries. The high prevalence of the various thalassemic genes in these populations is probably explained by a selective pressure exerted by malaria. Thalassemias are classified according to the type of chain which is affected: the two main categories consist of LX-and /?-thalassemias: each can be subdivided into several different subtypes.
Genetics of or-thalassemia The genetics genes (Higgs
of cc-thalassemia are most frequently explained by the deletion of a et al., 1989). Normal individuals have two CI genes (W/W) on each 129
130
Claude Poyart and Henri Wajcman
chromosome 16, which are named ct2 and xl, from 5’ to 3’. The a2 gene encodes two- to three-fold more protein than the xl gene. Carriers of cc-thalassemia have either three (-~/GYM)or two (--/w or -Z/-(X) r genes. These individuals having only two functional genes are now classified as being heterozygous for an a”-thalassemia or homozygous for an r+ -thalassemia, respectively. The a-thalassemic patients suffering from hemolytic anemia usually have an excess of free p chain tetramers (HbH) in their red blood cells (RBC) because of the deletion of three 9 genes (-l-x). The homozygous situation for a”-thalassemia leads to Bart’s hydrops fetalis syndrome responsible for fetal death ir2 utero. A consequence of these genetic exchanges is the existence of individuals clinically normal and having five or six genes. Deletions within the x gene cluster may be favored by the presence of many repetitive sequences, with a surprisingly high G+C content (Lauer et al., 1980). They may result either from reciprocal recombination between two chromosomes or from gene conversion involving the two genes of the same cluster. The region involving the two c( genes may be divided into homologous subsegments (X, Y and 2) and nonhomologous elements (I, II and III). Recombination between 2 segments results in cr+-thalassemic chromosomes having the ~~~~~ rightward deletion (Fig. 4). Recombination between the homologous X segment gives rise to the -a4.’ leftward deletion. In both cases, several subtypes may be distinguished according to the exact their point of recombination. Homozygotes for the - c(‘.’ deletion have theoretically two a2 genes missing but the amount of x-globin synthesized is higher than the 25% keep expected. Conversely, homozygotes for the - r3.7 deletion, which theoretically of the equivalent of two ct2 genes, express only about half of the normal quantity which involve cc-globin instead of the expected 75% (Bowden et al., 1987). Deletions
w
a2
a2
al
al enli-a 3.7
-a 3.7 ylal
a2
al
between the two a Fig. 4. A mispairing in the homologous regions followed by recombination genes leads to a deletional a-thalassemia. In this example the mechanism involving the 2 homology blocks that gives the -a “’ rightward deletion is shown. X, Y, Z and I, II, III are the homologous and non-homologous regions, respectively. The distance between the two X blocks and the two 2 blocks is 4.2 and 3.7 Kb, respectively.
Hemolytic Table 1. Some unstable
*: chain variants responsible
Variant
Structural
Hb Hb Hb Hb Hb Hb Hb Hb
r14(A12) Trp+Arg r29(BlO) Leu+Pro 238 or 39(C4) Thr+0 a59(Gll) Gly-+Asp a104(E8) Cys+Tyr a109(G16) Leu+Arg ~llO(G17) Leu+Asp x125(H8) Leu+Pro
Evanston Agrinio Taybe Adana Sallanches Suan Dok Petah Tikva Quong Sze
*Compound
heterozygote
131
Anemias Due to Hemoglobinopathies
modification
for r-tha12, **homozygote,
for non-deletional
% HbH
Gene Rightward x2 Xl xl a2 r2 ? x2 ***compound
thalassemias
r-tha12
heterozygote
Traces Traces* 0.5** Traces*** 2** 15*** 3*** 35*** for a-thall.
both the x.2 and the ctl genes are responsible for x0-thalassemia. Several types been described that may be distinguished by their length and starting point.
have
Many examples of a-thalassemic patients with HbH disease have been found in which one of the CI genes is present but not functional. The reason is the presence of a mutation(s) in a control region affecting the expression of this gene. Several examples are known of such non-deletional a-thalassemic genes. As shown in Table 1, the same picture may be produced by the presence of a mutation encoding for an unstable protein (Kan et a/., 1977).
Genetics of /I-thalassemia In the b-thalassemias the gene is almost always present. A great variety of mutations have been described that may affect any of the regions involved in the control of the expression of the gene (Huisman, 1992). When the expression of the gene is totally abolished, the defect is known as PO-thalassemia and when globin is still synthesized in small amounts as /3+-thalassemia. HbE is both a structurally abnormal hemoglobin and a /I+-thalassemia (Orkin, 1987). In this example the codon 26 located at the end of the first exon is both a missense (Glu-+Lys) and an alternative site for splicing. Usually no hemolytic anemia is observed in the heterozygous carriers of a fl-thalassemic gene, the red cell parameters showing only a mild hypochromia and microcytosis. In contrast, a severe hemolytic anemia with a high degree of bone marrow stimulation is observed in patients that are either homozygotes or compound heterozygotes for the two different /I-thalassemic genes. The severity of the hemolytic process may vary to a large extent from the classical Cooley anemia that requires blood transfusion and iron chelation for life, to that of thalassemia intermedia in which blood transfusion is usually not necessary. In patients that associate both fi-thalassemic and x-thalassemic genes, the biosynthesis of both kinds of chains is decreased and, as a result, a lower amount of unpaired subunits accumulates in the RBC which are still hypochromic but the hemolytic process is less severe. Conversely when a patient associates a p-thalassemia and a triplication of the r genes in one of his chromosome 16 the clinical picture may be a thalassemia intermedia.
132
Claude Poyart and Her-vi Wajcman
The genetic mechanism for G/?-thalassemias is a deletion involving totally or partially both 6 and p genes. In these patients the expression of the y genes is usually high and the small amount of adult Hb is partly compensated by an increased synthesis of fetal Hb. As a result the hemolytic anemia syndrome is mild as in /Y-thalassemias.
Molecular genetics of abnormal hemoglobins From a genetic point of view, abnormal Hbs and thalassemia inherited. From a clinical point of view this is less evident carriers of thalassemic genes and of several abnormal Hbs are the biological consequences of the abnormahty may be difficult
are both codominantly since the heterozygous healthy. In some cases to identify.
A4olecular genetics of sickle cell disease HbS is the most frequent abnormal hemoglobin found in populations of African origin but is also observed in Arab and Indian populations. HbS results from the substitution of a valine for a glutamic acid at position 6 of the fl chain. HbS has a multi-centric origin (Pagnier et al., 1984) and appeared independently in at least five locations. Heterozygous carriers for HbS are usually healthy and detected only by biological and clinical hemoglobin studies. Clinical manifestations of the abnormal hemoglobin are exceptional, except for kidney lesions characterized by interstitial papillary necrosis (Lantz et al., 1995). Conversely, patients suffering from homozygote sickle cell disease exhibit a severe hemolytic anemia and varying secondary complications affecting all the organs by thrombo-embolism. This is also the situation for compound heterozygotes for HbS and a J?-thalassemic gene, or for individuals carrying in addition to HbS an additional mutation that enhances the polymerization processes of sickle cell Hb (Serjeant, 1985). The presence of various genetic factors that stimulate the production of fetal Hb (HPFH-like genes) leads to a sickle cell disease that is better tolerated than others. Five factors have been shown to influence the high variation of HbF levels in sickle cell anemia: age, sex, the IX globin gene number, ,O globin haplotypes and mostly an X-linked factor that regulates the production of HbF-containing erythrocytes (Chang et al., 1995). Since HbS is a frequent abnormality, several examples have been identified in which another genetic event, such as a second globin point mutation or a crossover with a gene carrying another gene, occurs in the same gene. This situation has been well documented in the case of HbC Harlem and HbS Antilles. Both variants carry the substitution of HbS. In the first case, it is associated with the mutation of Hb Korle Bu which possesses an anti-sickling effect (Bookchin et al., 1967). In the second case, the /36 Glu-+Val mutation is associated with the substitution of an Ile for a Val at position p23 (BS), which enhances HbS polymerization (Montplaisir et al., 1986). Many Hb variants, like HbS, are well tolerated in the heterozygous form but induce in homozygous carriers a mild hemolytic anemia, such as encountered HbC, which cnjstallizes within the red blood cells.
may in
Hemolytic Anemias Due to Hemoglobinopathies
133
Molecular genetics of hemolytic anemia due to unstable hemoglobins By contrast, unstable abnormal Hbs behave frequently as hereditary dominant. These Hbs have a structurally abnormal chain resulting in a molecular instability which causes CHBHA (Bunn and Forget, 1986). The heterozygous carriers suffer from a hemolytic anemia, the severity of which depends on the structural abnormality induced by the mutation (Ohba, 1990). In some cases, the anemia is very mild but in other cases is life threatening requiring frequent blood transfusions. According to the International Hemoglobin Information Center (199.5), approximately 200 different mutations have been identified as responsible for unstable Hbs.
Unstable Hbs result from private mutations limited to a few individuals or to a small number of families. Therefore, de nouo cases are not exceptional. Abnormal Hbs with identical pri.mary sequence structural abnormalities, as for example in Hb Koln [/I 98 (FG5) Val+Met], have been found in several unrelated patients. This is likely to be a consequence of the presence of hotspots for mutations. The frequency of mutations involving CpG dinucleotides seems to be higher than random (Perutz, 1990). The high number of de nouo cases of unstable well-characterized Hbs variants may, however, be biased relative to other Hb variants, by the fact that the Hb status of patients suffering from a hemolytic anemia is thoroughly investigated. The availability of sophisticated methods helps considerably to identify mutations that may otherwise behave as neutral with routine electrophoretic tests. In other cases, several siblings from apparently normal parents are affected; this may be explained by a germline mosaicism due to a somatic mutation having occurred at an early stage of the embryologic development of one of the parents (Wajcman et al., in press). Unstable Hbs may also result from several mechanisms, such as the simultaneous presence of two mutations in the same gene or from the addition or deletion of short sequences. These more complex genetic events may be explained by the presence of DNA sequences having a sufficient degree of homology to allow mispairing followed by breakage and incomplete reparation (Cooper and Krawczak, 1991; Krawczak and Cooper, 1991).
Unstable hemoglobins and dominant thalassemias When the instability of the CX-or /I-globin chains is very high, with a half-life of only a few minutes or hours, the molecule is denatured and lost in the red cell precursors of the bone marrow and the clinical expression becomes similar to that of a thalassemia intermedia. The main difference with a thalassemic syndrome consists of the genetic inheritance which follows a clinically dominant mode. These Hbs are indeed often classified as dominant thalassemias. In the cases where the half-life is slightly longer, all the abnormal Hb may be eliminated in the spleen and all the tests for an unstable Hb can be negative in the peripheral blood. In some of these patients, the abnormal Hb has been detected only after splenectomy.
134
Claude Poyart and Henri Wajcman
Pathophysiological Mechanisms The pathophysiology of hemolysis shares a common mechanism in the various hemoglobinopathies. In unstable Hbs, the structurally abnormal Hbs form precipitates that are bound by the membrane skeleton, decreasing the cell deformability and revealing abnormal epitopes favoring lysis. In the case of thalassemia, the free subunits, which are in excess, behave like an unstable Hb. In sickle cell disease, the polymerized molecules of deoxy HbS alter the shape of the cell and the structure of the membrane, leading to membrane loss and ultimately to cell destruction. In all these cases, the hemolysis is intra- and mostly extravascular. The patients suffer from a chronic hemolysis. In normal conditions and due to efficient bone marrow compensation, the anemia is well tolerated.
Genera/ mechanisms
for extravascular hemolysis
Heinz body formation Heinz bodies are the products of Hb degradation. The precipitated material is made of hemichromes, which are low spin derivatives of ferric Hb (Rachmilewitz, 1974). In some of them, the sixth coordination position of the heme is occupied by a ligand that may be a hydroxyl, a water molecule, a protonated or an unprotonated histidyl provided by the globin. Hemichromes are generated when the heme dissociates from its normal position in the heme pocket and rebinds elsewhere in the globin. Denatured hemoglobin binds with extremely high affinity to proteins of the erythrocyte membrane, principally to the cytoplasmic portion of band 3 (also named erythrocyte anion exchange channel) (Waugh and Low, 1985). This causes band 3 to aggregate, forming clusters that are responsible for the appearance of senescence antigens on the surface of the cell. These epitopes are recognized by macrophages. Deposits of immunoglobulins specific to these abnormal epitopes can be responsible for some degree of immunological lysis of the cell in conjunction with complement. Heinz bodies also generate many oxidant agents (superoxide, peroxide or hydroxyl radicals) that damage the proteins and the lipids of the membranes. The presence of Heinz bodies decreases the deformability of the erythrocyte and enhances its fragility; two factors leading to the destruction of the cell in the spleen that will be considered below.
Spleen and hemolysis The extravascular hemolytic process is mediated by the macrophages of the bone marrow, the liver and the spleen. Severe unstable Hbs, in which the subunits are not able to assemble into soluble tetramers, result in a high degree of hemolysis in the bone marrow. In contrast when the tetramers become unstable following oxidative stress, the destruction of the RBCs occurs mainly in the spleen. Normally, all the circulating RBCs are controlled by the spleen allowing the removal of aged or damaged cells (Chen and Weiss, 1973). Only the erythrocytes, which have a normal deformability, may pass through the filter of the spleen without damage. Poorly deformable cells or cells containing precipitated materials are not able to pass
Hemolytic
Anemias Due to Hemoglobinopathies
135
safely between cordal macrophages that recognize abnormal membrane epitopes. These cells also have difficulties passing through the fenestrations of the basement membrane of the sinus and between the endothelial cells. The spleen, in addition to its culling function, e.g. the removal of abnormal and deleterious cells, has a pitting function that consists of removing inclusion bodies formed within the cells. When a erythrocyte containing inclusion bodies attempts to pass through the sinusoidal filter, the inclusions accumulate in the last part of the cell which can no longer enter the sinus. This part of the cell which has lost its deformability is then removed by fragmentation. The fragments released will be digested by the macrophages while the remainder of the RBC returns in the circulating blood. The damaged cells having lost some membrane material are more fragile than normal and will soon become a target for destruction by macrophages. They may also be culled by the spleen in a subsequent passage.
Hemolysis in unstable hemoglobins Substitutions in the primary sequence of the Hb chains can result in a subunit that is unstable and tends to precipitate. These mutations usually affect a functional region critical for the stability of the molecule: examples are the heme pocket, the intersubunit contact areas in the dimers or tetramers or the interior of the globin. Instability may also result from a large structural perturbation due to the disruption * of a helical structure or to an amino acid deletion (Fermi and Perutz, 1981). In the heme pocket different types of structural abnormalities have been described. When the structural abnormality involves the proximal side of the heme, the strength of the bond between the heme and the F helix of the globin is decreased. This is illustrated by several variants, such as Hb Boras [fl88(F4) Leu-+Arg] (Hollender et al., 1969), Hb Bristol [/?67(Ell) Val+Asp] (Steadman et al., 1970) or Hb Olmsted [p141(H19) Leu-*Arg] (Lorkin et al., 1975). Some examples are also known in which the proximal histidine (F8) is replaced by a residue that cannot bind the heme. This leads to the formation of unstable tetramers, named semi-hemoglobins, in which only the normal chains carry a functional heme group. Typical examples of this pathology are Hb Redondo [p92 (F8)His+Asn] (Wajcman et al., 1991) or Hb Saint Etienne [fl92 (F8) His-tGln] (Beuzard et al., 1972). In Hb Redondo, the instability is enhanced by the deamidation of the asparagine residue replacing the F8 histidine, which may lead to some cleavage of the protein backbone. Conversely, when the structural modification is localized on the distal side of the heme, the aperture of the heme pocket may be increased, favoring entry of water which increases the rate of oxidation. Classical examples are provided by Hb Zurich [p63 (E7)His+Arg] (Muller and Kingma, 1961) and Hb Bic&tre [p63 (E7)His-+Pro] (Wajcman et al., 1976) in which the distal histidine is replaced by an arginine and a proline, respectively. In Hb Bic&tre, the rate for autooxidation of the abnormal chains is increased and the tetramers behave as p valency hybrids in which the a chains bind oxygen but the p chains are in the metHb form. Hb Zurich has both an increased autooxidation rate and a much higher than normal affinity for carbon monoxide. Carriers of Hb Zurich that are smokers therefore display a high level of HbCO which
136
Claude Poyart and Henri Wajcman
is much more stable than the metHb. The CHBHA of these patients is therefore very atypical: Heinz bodies in the red cells are rare and the Hb level is normal or even increased due to a high oxygen affinity. Another group of mutations involves residues that participate in subunit interactions. In the tetrameric structure of Hb, the interface between the c( and p subunit (crij3i contact) of each dimer is relatively rigid. Mutations occurring in this region lead to instability because the tetrameric assembly dissociates into dimers and then monomers. These free subunits uncoil, have a high autooxidation rate (16 times that of native HbA) and finally form hemichromes that precipitate. Examples are provided by many variants among which Hb Philly l-835 (Cl)Tyr+Phe] (Rieder et al., 1969) in the p chains or Hb Taybe (Galacteros et al., 1994) in the c1 chains. In the case of a chain variants the unstable Hb may be responsible for the presence, in addition to traces of HbH (but to a much lower extent than in cc-thalassemia) of an artifactual fi-thalassemic-like biosynthetic ratio. The hypothesis of a disturbed association between subunits may conciliate these paradoxical observations. The introduction of a proline, or sometimes of a glycine, in the middle of a helix disrupts the secondary structure and leads to the instability of the Hb molecule. This mechanism is one of the most frequently observed. Mutations that modify the folding interactions between the different CI helical regions or which permit the introduction of additional water molecules which are not present normally inside the Hb, may also modify the solubility of the molecule and lead to instability and the subsequent oxidation and precipitation. Some Hbs are designated as hyperunstable because they are destroyed so rapidly that they are barely detectable or undetectable in the hemolysate. These Hbs usually result from mutations localized in the third exon, in regions coding for the ccl/?1 contact. As shown in Table 2, they include point mutations, insertions or deletions of residues or frameshift. In the case of the double mutant Hb Medecine Lake [P32(B14) Leu-+Gln; /398(FG5) Val-+Met] (Coleman et al., 1995) the same globin chain carries two substitutions that even when present alone are responsible for CHBHA. Some of these hyperunstable variants may be detected after splenectomy in the hemolysate.
Hemolysis in thalassemia The reduced synthesis of one of the globin chains results in an overall deficit in Hb accumulation and to the presence of a large excess of the non-affected globin chain. These free subunits form unstable aggregates that precipitate within the cell leading to the above described membrane disorders.
P-thalassemia In P-thalassemia, the inclusions bodies contain aggregated a-chains. They are formed of the bone marrow, leading to ineffective in the etythrocyte precursors erythropoiesis. Usually, these Heinz bodies have disappeared from the cytosol of the activity is high in the erythrocyte circulating erythrocytes since the protease By contrast, they are often observed in bone marrow smears. In this precursors.
137
Hemolytic Anemias Due to Hemoglobinopathies regard, thalassemic syndromes differ from unstable formation occurs most frequently in the peripheral oxidative stress.
Hbs blood
in which Heinz when submitted
body to an
cl-thalassemia In ol-thalassemias, the excess of P-chains form tetramers (Hb H) which are more stable than the free LXchains. As a consequence, ineffective erythropoiesis and bone marrow destruction of the erythrocyte precursor is less marked. The precipitation of Hb H proceeds more gradually and occurs in the peripheral blood rather than in the precursors of the bone marrow. The pathophysiological mechanism of hemolysis, in which the spleen plays a crucial function, becomes, in this case, very similar to that of unstable Hbs.
Hemolysis in sickle cell disease Hb S polymerizes when the cell is submitted to reduced oxygen tension, forming long fibers that modify the shape of the red cells. As long as the membrane of the cell has
Table 2. Mechanism
Substitution involving one residue Chesterfield Cagliari Terre Haute Showa Yakushiji Durham NC Houston Presence of two substitutions Medicine Lake
for hyperunstable
variants affecting the /I’chain
fl28 Leu+Arg /3 60 Val+Glu j 106 Leu-rArg /I 110 Leu-+Pro b 114 Leu+Pro /I127 Ala-Pro
in the same chain /I 32 Leu-+Gln,
fl98 Val+Met
Deletion or addition of one or several codons Korea /3 33 or 34 (Val+O) Gunma codons 127-128 (Gin-Ala+Pro) 0 fi 134-137 (- 12, +6) (Val-Ala-Gly-Val-Gly-Arg) Koriyama /? 95(+Leu-His-Cys-Asp-Lys-) 96 Early chain termination 0 0 Frameshift by deletion Agnana 0 Manhattan Geneva Makabe Kohn Kaen Vercelli 0
codon 121 (GAA-+TAA: codon 127 (CAG+TAG:
Glu-+Term) Gln-rTerm)
or addition of one or several nucleotides codon 94 (+TG) + 156 residues long chain codons 106-107 (+G) codon 109 (-G) -+ 156 residues long chain codon 114 (-CT, +G) + 156 residues long chain codon 123 (-A) + 156 residues long chain codons 123-125 (-ACCCCACC)+156 residues long chain codon 126 (-T) +156 residues long chain codons 128 (-4,+5, -11) + 153 residues long chain
138
Claude Poyart and Henri Wajcman
not been too severely injured, sickling is reversible upon oxygenation. After several oxy-deoxy cycles, the membrane alterations accumulate and sickling becomes an irreversible process. The irreversibly sickled cells are dehydrated dense cells containing a large amount of polymerized Hb S. Their number correlates well with the hemolytic rate and the spleen size. In normal red cells the phospholipids of the membrane are asymmetrically distributed: amino phospholipids (phosphatidyl serine and phosphatidyl ethanolamine) the inner layer and choline-phospholipids are mainly in (phosphatidylcholine and sphingomyelin) in the external one. As a consequence of the injuries produced by the Hb S polymers, this asymmetric composition is barely maintained. This abnormal lipid partitioning leads to several consequences among which are an enhancement of the procoagulant activity and an increased adhesion to the macrophages. Hb S also leads to the formation of micro-Heinz bodies, which are much smaller than those observed in unstable Hbs. These inclusion bodies generate potent oxidants which alter the structure of the membrane. In addition, they bind with a high affinity to the cytoplasmic region of band 3 causing it to aggregate. The distribution and nature of the surface epitopes of the RBC is therefore modified. This process has been involved in the formation of senescence antigens leading to hemolysis.
Clinical Picture An extensive literature is already available on the clinical presentation of thalassemias and sickle cell disease and it is out of the scope of this short review to develop these points. Therefore, we will limit this chapter to the clinical picture of unstable Hbs.
Historical The first case of congenital hemolytic anemia due to an unstable Hb has been reported by Cathie (1952). It concerned a baby that was 10 months old when diagnosed. The child presented jaundice, splenomegaly and emission of dark urines. This observation differed from familial spherocytosis because the hematological disorders did not disappear after splenectomy. The biochemical identification of the molecular abnormality was discovered only 18 years later when it was shown that the baby carried an abnormal Hb (named Hb Bristol), in which the valine residue at position /?67 (El 1) was replaced by an aspartic acid (Steadman et al., 1970).
Biological
diagnosis of an unstable Hb
The discovery of Heinz bodies in blood smears after supra Hb. The presence suggest the presence of an unstable nevertheless not pathognomonic of an unstable Hb and several red cell enzymopathies, are also known to produce them.
vital staining usually of Heinz bodies is mechanisms, such as
The definition of an unstable Hb is a biochemical one: it means that the solubility of the Hb molecule is reduced as compared to that of Hb A (Huisman and Jonxis, 1977).
Hemolytic Anemias Due to Hemoglobinopathies
139
Thus, historically, an unstable Hb is a molecule that precipitates when incubated for 1 hour at 50°C. In these conditions only the severe unstable Hb are detected. More sensitive tests are now available which lead to positive results for less unstable molecules. One of the most widely used consists of incubating the lysate at 37°C in the presence of 17% isopropanol during a length of time insufficient to precipitate Hb A (Carrel and Kay, 1972). Another accurate method is to record the rate of denaturation of the Hb molecule under standardized conditions of heme concentration and at a higher temperature (65°C). The test should be done on a hemoglobin solution previously equilibrated under 1 atm oxygen for 20-30 min in order to test fully liganded tetramers. When a Hb is very unstable, its amount in the circulating blood may be too low to lead to a clear precipitate and the instability tests may be erroneously considered as negative. The same situation may occur for an unstable Hb that is encoded by a gene expressed at a low level such as the xl gene (Galacteros et al., 1994). Unstable Hbs that have a half-life of only a few hours may be detected by chain biosynthesis measurements. When the reticulocytes of these patients are incubated during a short time period, 1 hour or less, in the presence of a labeled amino acid, the abnormal chain which is synthesized at a normal rate may be demonstrated.
Clinical severity of unstable hemoglobins The clinical disorders and, therefore, several
are directly proportional to the instability of the Hb molecule degrees of severity may be recognized, as shown in Table 3.
In many cases the slightly unstable Hb molecule variants are detected by in vitro studies; these variants are without clinical consequences unless they are associated with another red cell or membrane abnormality or submitted to a strong oxidative stress. In the case of moderately unstable Hbs, the anemia is the most frequently well compensated. The patient may nevertheless suffer from hemolytic crises during infectious episodes likely to be related to pyrexia or transient acidosis. In some cases, according to the structural abnormality involved, an oxidative drug may be the trigger mechanism of a hemolytic crisis. Emission of dark urines is characteristic but not
Table 3. Chnical manifestations
PI PII [III1 F
IYI
of unstable
Hbs (from Ohba, 1990)
Life long severe hemolytic anemia Life long mild to moderate hemolytic anemia Mild hemolytic anemia with slight jaundice Positive test for instability only found during in vitro studies “Dominantly inherited /?-thalassemia“ Recessively inherited r-gene disorders with Hb H traces
140
Claude Poyart and Henri Wajcman
always noticed. In the steady state, the patient does not require except for supportive and preventive measures (administration prevention of infection, avoidance of oxidative drugs).
specific therapy of folic acid,
Frequently the decreased stability is not the only abnormal feature of an unstable Hb molecule. The structural modification may affect a region which is not only crucial for stability but also for oxygen binding. Modified oxygen binding properties are therefore frequently associated with instability. When the affinity for oxygen is increased the oxygen delivery to the tissues is impaired. This leads to a stimulation of erythropoiesis which may increase the Hb level to values close to normal, such a patient will only have an apparently well compensated anemia. In contrast, unstable Hbs with decreased oxygen affinity may appear to be more severe. In the steady state these patients have a significantly lower Hb level than the previous ones and this value may drastically drop during hemolytic crises. Severe hemolytic disorders may require splenectomy and the patients then become submitted to specific complications among which are serious thrombo-embolic which are a major cause of episodes (Beutler et al., 1974). These complications, morbidity in sickle cell disease, have also been reported in thalassemia intermedia and other hemoglobinopathies. Many abnormalities of the RBC, that are present in carriers of unstable hemoglobins, have been implicated as causes of an increased adhesiveness. Membrane sialic acid abnormalities, oxidative membrane damage, loss of membrane phospholipid asymmetry, and binding to various plasma adhesive proteins (fibrinogen, fibronectin, von Willebrand factor) are among these factors. Owing to the exteriorization of procoagulant phospholipids in the RBC membranes, thrombin generation and fibrin formation appear also to be increased whereas antithrombin III, protein C and free protein S are reduced (Francis and Johnson, 1991). In contrast, other variants lead to very severe hemolytic disorders with a red cell life limited to a few days or even to a few hours. These hyperunstable Hb variants are destroyed in the erythroblasts of the bone marrow and result in a syndrome having clinical manifestations similar to those observed in thalassemia intermedia (Adams and Coleman, 1990; Thein et al., 1990; Thein, 1992). All the intermediary forms between these two extremes have been described. Unstable variants due to a structural modification carried by the a-chain have biological and clinical expression varying considerably from one case to another. This may be explained by the fact that or-chains are encoded by two genes. Since the a2 gene encodes for a higher level of mRNA than the cxl gene, a greater impact of an a2 locus mutation is predicted, in comparison to mutations of the al gene. In addition, according to the mechanism of instability involved, an abnormality of the ~2 locus may lead either to a ‘classical unstable Hb syndrome’ or to an cc-thalassemia like phenotype when the Hb is destroyed in the bone marrow. In some ‘thalassemic (Goossens et at., 1981), the variants’, such as Hb Quong Sze [a125 (H8) Leu +Pro] mutated chain is destroyed before assembling with the partner subunit, leading to a pure a-tha12 including some degree of ineffective erythropoiesis (Table 1). In other variants, the clinical picture results from a mixture of two features: (1) an increased erythropoiesis and (2) an early precipitation of the abnormal hemoglobin during RBC
Hemolytic Anemias Due to Hemoglobinopathies
141
maturation. In some cases, such as Hb Questembert [a131 (H14) Ser+Pro] (Wajcman et al., 1993) or Hb Ann Arbor [a80 (Fl) Leu+Arg] (Adams et al., 1972) the structural abnormality may result in a disturbed protein folding and subunit assembly and in paradoxical biosynthetic ratio.
Biological Bases for Therapy In spite of the profound understanding of the molecular pathogenesis of hemoglobinopathies, no efficient specific treatments are available. Nevertheless, recent advances in transfusional programs, in pharmacology, in allogeneic bone marrow transplantation and in molecular genetics offer considerable promises in preventing or delaying most of the complications associated with hemoglobinopathies (Rodgers et al., 1994).
Progress in iron chelation therapy Striking improvement in life expectancy has been obtained in the treatment of thalassemia by hypertransfusion programs when associated with an effective prevention from the toxic effects of iron overload. Up to now the only therapeutically available iron chelating agent is deferoxamine. It needs to be administrated by parenteral mode and the prognosis of a patient submitted to nightly subcutaneous deferoxamine perfusion has been considerably improved. Also, very efficient programs, using pumps for continuous ambulatory intravenous perfusion over several days per month have been designed. An orally active chelator, L- 1 (1,2-dimethyl-3-hydroxypyrid-4-one) has been recently proposed for patients unable to comply with parenteral therapy. A large controlled multicentric study has recently been initiated to determine the efficiency and the toxicity of this drug (Olivieri et al., 1995).
Allogeneic
bone marrow transplantation
Since the initial report in 1982 of a bone marrow transplantation in a thalassemic patient, several hundreds of thalassemic patients, all over the world, have been cured by this treatment. Survival rates have clearly improved with more effective prevention of infections, of graft failure and of graft versus-host-disease. The experience is much more limited with sickle cell disease. The risk of complication of a bone marrow transplant seems now to be sufficiently low to consider such a treatment for prevention of long term morbidity in some selected patients suffering from serious forms of hemoglobinopathies. The inclusion criteria for sickle cell patients to benefit from this therapy have been recently determined (Bernaudin et al., 1995).
Modulation
of globin gene expression
Since Hb F interferes with the polymerization of deoxyHb S in sickle cell disease and compensates for the unbalanced chain biosynthesis of /&thalassemia, several agents
142
Claude Poyart and Henri Wajcman
have been evaluated for their effect in the augmentation of synthesis of Hb F in these hemoglobinopathies. In sickle cell disease, a significant effect of Hb F requires both a total Hb F level of at least 20% with a distribution over 75% of the RBC. To achieve this goal several agents such as cytotoxic agents (5azacytidine or hydroxyurea), hematopoietic growth factors (cytokines, erythropoietin), or short chain fatty acids (arginine butyrate) have been proposed. Hydroxyurea is the least toxic of the cytotoxic agents. It has been used widely for patients with sickle cell disease and it has been demonstrated that most patients respond to this drug by doubling their baseline level of Hb F and F-reticulocyte count (Charache et al., 1992). Long-term therapy seems to decrease the frequency and severity of pain crisis and to improve quality of life of the patients. Results concerning /3-thalassemic patients are less clear. Among the hematopoietic growth factors only recombinant human erythropoietin has been reported in clinical trials (Rachmilewitz et al., 1991). It was demonstrated to increase the percentage of F-reticulocytes and Hb F level in sickle cell patients when administered at high doses along with supplemental iron. Also, erythropoietin exerts an additive effect when administered with hydroxyurea. Clinical trials are underway. Attention has recently been focused on butyrate analogs and other short chain fatty acids. These molecules have been found to induce hematopoietic cell differentiation and to increase Hb F level. Clinical trials with phenylbutyrate alone or in combination with other agents are underway in sickle cell and in thalassemic patients (Perrine et al., 1993).
Genetic treatment Hemoglobinopathies are good candidate diseases for gene therapy since they could be cured by inserting an efficient fl-globin gene in the progenitor cells (Sadelain, 1995). This strategy awaits the solution of several biological problems which are out of the scope of this review.