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 of cc-thalassemia are most frequently explained by the deletion of a

genes (Higgs 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 non-
homologous 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
point of recombination. Homozygotes for the - c(‘.’ deletion have theoretically their
two a2 genes missing but the amount of x-globin synthesized is higher than the 25%
expected. Conversely, homozygotes for the - r3.7 deletion, which theoretically keep
the equivalent of two ct2 genes, express only about half of the normal quantity of
cc-globin instead of the expected 75% (Bowden et al., 1987). Deletions which involve

w a2 al

a2 al
enli-a 3.7

-a 3.7
ylal a2 al

Fig. 4. A mispairing in the homologous regions followed by recombination between the two a
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 Anemias Due to Hemoglobinopathies 131

Table 1. Some unstable *: chain variants responsible for non-deletional thalassemias

Variant Structural modification Gene % HbH

Hb Evanston r14(A12) Trp+Arg Rightward r-tha12 Traces

Hb Agrinio r29(BlO) Leu+Pro x2 Traces*
Hb Taybe 238 or 39(C4) Thr+0 Xl 0.5**
Hb Adana a59(Gll) Gly-+Asp xl Traces***
Hb Sallanches a104(E8) Cys+Tyr a2 2**
Hb Suan Dok a109(G16) Leu+Arg r2 15***
Hb Petah Tikva ~llO(G17) Leu+Asp ? 3***
Hb Quong Sze x125(H8) Leu+Pro x2 35***

*Compound heterozygote for r-tha12, **homozygote, ***compound heterozygote for a-thall.

both the x.2 and the ctl genes are responsible for x0-thalassemia. Several types have
been described that may be distinguished by their length and starting point.

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 are both codominantly
inherited. From a clinical point of view this is less evident since the heterozygous
carriers of thalassemic genes and of several abnormal Hbs are healthy. In some cases
the biological consequences of the abnormahty may be difficult 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 may
induce in homozygous carriers a mild hemolytic anemia, such as encountered in
HbC, which cnjstallizes within the red blood cells.
 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
in the etythrocyte precursors of the bone marrow, leading to ineffective
erythropoiesis. Usually, these Heinz bodies have disappeared from the cytosol of the
circulating erythrocytes since the protease activity is high in the erythrocyte
precursors. By contrast, they are often observed in bone marrow smears. In this
 Hemolytic Anemias Due to Hemoglobinopathies 137

regard, thalassemic syndromes differ from unstable Hbs in which Heinz body
formation occurs most frequently in the peripheral blood when submitted to an
oxidative stress.

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 for hyperunstable variants affecting the /I’chain

Substitution involving one residue

Chesterfield fl28 Leu+Arg
Cagliari /3 60 Val+Glu
Terre Haute j 106 Leu-rArg
Showa Yakushiji /I 110 Leu-+Pro
Durham NC b 114 Leu+Pro
Houston /I127 Ala-Pro
Presence of two substitutions in the same chain
Medicine Lake /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 codon 121 (GAA-+TAA: Glu-+Term)
0 codon 127 (CAG+TAG: Gln-rTerm)
Frameshift by deletion or addition of one or several nucleotides
Agnana codon 94 (+TG) + 156 residues long chain
0 codons 106-107 (+G)
Manhattan codon 109 (-G) -+ 156 residues long chain
Geneva codon 114 (-CT, +G) + 156 residues long chain
Makabe codon 123 (-A) + 156 residues long chain
Kohn Kaen codons 123-125 (-ACCCCACC)+156 residues long chain
Vercelli codon 126 (-T) +156 residues long chain
0 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) are mainly in the inner layer and choline-phospholipids
(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 vital staining usually
suggest the presence of an unstable Hb. The presence of Heinz bodies is
nevertheless not pathognomonic of an unstable Hb and several mechanisms, such as
red cell enzymopathies, are also known to produce them.

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 are directly proportional to the instability of the Hb molecule
and, therefore, several 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 of unstable Hbs (from Ohba, 1990)

PI Life long severe hemolytic anemia

PII Life long mild to moderate hemolytic anemia
[III1 Mild hemolytic anemia with slight jaundice
Positive test for instability only found during in vitro studies
F “Dominantly inherited /?-thalassemia“
IYI 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 specific therapy
except for supportive and preventive measures (administration of folic acid,
prevention of infection, avoidance of oxidative drugs).

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
episodes (Beutler et al., 1974). These complications, which are a major cause of
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
variants’, such as Hb Quong Sze [a125 (H8) Leu +Pro] (Goossens et at., 1981), the
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.