Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2141-2146
Is Hemoglobin Instability Important in the Interaction Between
Hemoglobin E and 
Thalassemia?
By
D.C. Rees,
J.B. Clegg, and
D.J. Weatherall
From the MRC Molecular Haematology Unit, Institute of Molecular
Medicine, University of Oxford, The John Radcliffe, Headington, Oxford,
UK.
 |
ABSTRACT |
Hemoglobin E (HbE;
2
226glu-lys), globally the
commonest hemoglobin variant, is synthesized at a slightly reduced rate
and has a homozygous phenotype similar to heterozygous thalassemia.
Yet, when it is inherited together with a thalassemia allele, the
resulting condition, HbE/ thalassemia, is sometimes characterized by
a severe, transfusion-dependent thalassemia major. The severity of this
interaction has not been explained. We have explored the possibility
that it may reflect the instability of HbE consequent upon globin chain
imbalance imposed by the thalassemia allele. Time-course and
pulse-chase globin chain synthesis studies at 37°C on peripheral
blood and bone marrow suggest that hemoglobin instability is not
significant in steady-state HbE/ thalassemia; this is confirmed by
density-gradient centrifugation studies that show no decrease in HbE
levels relative to HbA as HbE/+ thalassemia red blood
cells age. Globin binding to membranes was assessed and only globin
chains were found, in contrast to other unstable hemoglobins in which
both and chains were present. However, in experiments performed
on blood from HbE/ thalassemics in the temperature range 39°C to
41°C, there was evidence of instability of HbE, a finding that was
also observed in homozygous HbE. These findings suggest that the
phenotype of HbE/ thalassemia is primarily the result of the
interaction of two thalassemia alleles; however, hemoglobin
instability may be important during febrile episodes, contributing to
worsening anemia.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
HEMOGLOBIN E (HbE;
2226glu-lys) is the commonest
hemoglobin variant, most prevalent in the rapidly expanding populations
of Southeast Asia1,2 but being encountered with increasing
frequency in the immigrant peoples of Europe, North America, and
Australia. Although HbE was first identified in 1954,3
there are still uncertainties about many aspects of its
pathophysiology. Both heterozygotes and homozygotes are asymptomatic,
minimally anemic, and have microcytic and hypochromic red blood cells.
The latter is explained by the thalassemic nature of the
E allele,4 which reflects the activation of
a cryptic donor splice site by the GAG to AAG mutation in exon 1 of the
globin gene.5 However, when the E allele
interacts with a thalassemia mutation in the compound heterozygous
state, a variable and often severe anemia is produced, with hemoglobin
levels ranging from 3 to 11 g/dL.6 The severity of this
interaction with thalassemia seems disproportionate to the very
mild defect in globin chain synthesis associated with HbE; HbE
homozygotes have the hemoglobin and globin chain synthesis
characteristics of thalassemia trait and have no elevation of HbF.
A clue to a possible mechanism involved in the interaction of HbE and
thalassemia lies in the observation that HbE is oxidatively unstable in vitro7,8 and that drug-induced oxidation of HbE is faster than that of hemoglobins S, A, and F.9 It is
thought that this increased instability results from interference with the 11 interface,9,10 resulting in an increased rate
of dissociation of the HbE molecule into unstable dimers. The
significance of this instability in vivo is unclear, but we have
recently described a family in which HbE interacts with a red blood
cell enzymopathy, pyrimidine 5
nucleotidase deficiency, to produce an
unexpectedly severe anemia11; the basis for this
interaction was shown to be increased HbE instability, presumably
resulting from the intracellular oxidative stress associated with the
enzymopathy. Using similar methods, we have explored the possible role
that hemoglobin instability plays in the interaction between HbE and
thalassemia.
| |
MATERIALS AND METHODS |
Subjects.
Blood was obtained from individuals resident in the UK who were known
to have HbE/ thalassemia; none of them had received blood
transfusions in the preceding 6 months. Control samples were collected
from relatives of the HbE/ thalassemics, thalassemia intermedia
patients (not involving HbE), and patients with known hemoglobin
instability. Informed consent was obtained in each case.
Hematologic analysis.
Blood was collected into EDTA. Full blood counts were performed using
an automated counter. Blood films, reticulocyte stains, and hemoglobin
isoelectric focusing were performed using standard techniques.12 These methods were used to confirm the
diagnosis, assess whether ° or + thalassemia was
present, and confirm the untransfused status.
DNA analysis.
DNA was isolated from peripheral blood leukocytes.13 The
nature of the thalassemia mutation was determined by
allele-specific polymerase chain reaction (PCR) amplification. Thalassemia was screened for by digesting DNA with Bgl II,
Southern blotting, and hybridization with a 
globin-specific probe.
Triplicated globin genes were screened for with BamHI
digestion and globin-specific probe hybridization.14
Globin-chain biosynthesis.
Approximately 20 mL of venous blood was taken into heparin and kept on
ice until analyzed, a maximum of 2 hours. The cells were washed three
times in cold reticulocyte saline (0.13 mol/L NaCl, 0.005 mol/L KCl,
0.007 mol/L MgCl2) before ultracentrifugation at
100,000g to concentrate the reticulocytes. White blood cells were removed from the top 0.6 mL of concentrated red blood cells using
-cellulose and microcrystalline cellulose (Sigmacell 50; Sigma
Chemical Co, Poole, Dorset, UK).15 This reticulocyte-rich fraction was added to a plasma-based incubation medium and, after 10 minutes of preincubation, 200 µCi of L-[4,5-3H] leucine
(Amersham International plc, Little Chalfont, Bucks, UK) was added and
the incubation was continued for 2 hours.16 Incubation
temperatures of 37°C, 39°C, and 41°C were used. A bone marrow aspirate sample taken for diagnostic reasons from an HbE/ thalassemic was also studied. Processing was similar apart from EDTA
being used as an anticoagulant; the white blood cells were not removed.
In time-course studies, aliquots were removed from the mixture at
various intervals from the start of the incubation. Each fraction was
washed with cold reticulocyte saline and flash frozen before conversion
to globin by acid-acetone precipitation. The globin chains were
separated by cation exchange chromatography; a linear two-chambered
gradient, producing 70 fractions, was used for HbA and a convex,
three-chambered gradient, producing 120 fractions, was used for samples
containing HbE. The radioactive incorporation into each fraction was
measured by scintillation counting, and the area under the curve of
each peak was calculated to measure the relative rates of synthesis of
the various chains over a particular time period.16
Specific activities (SA) of the globin chains were measured from each
separation, after dialysis of the peak fractions against 0.5% formic
acid. Hemoglobin stability was assessed by considering the rate of
increase of specific activity over 2 hours. If the rate of globin chain
synthesis is constant and the hemoglobin formed stable, then the SA
should increase linearly with time; this was expressed as the ratio of
SA increase in the first hour to that in the second hour. If the
hemoglobin is stable, then this ratio is approximately 1; if the
hemoglobin is unstable, newly synthesized globin chains are
proteolysed, the SA increases less as the incubation progresses, and
the ratio increases exponentially.
Pulse-chase studies were also performed. After incubation with
L-[4,5-3H] leucine for 10 minutes, the reticulocytes were
washed four times in cold reticulocyte saline to remove as much
unincorporated radioactivity as possible; the reticulocytes were then
added to fresh incubation medium with cold leucine replacing the
radiolabeled and the incubation continued. At various time intervals,
aliquots were removed, washed, frozen, and converted to globin. Globin chain separation, scintillation counting, and specific activities were
performed as described earlier.
Membrane studies.
The binding of radiolabeled globin to membranes was also studied using
a modification of Dodge's method to produce Hb-free ghosts.17,18 Time-course and pulse-chase incubations were
performed, the cells were washed, and the aliquots were lysed with 20 mOsm phosphate buffer, pH 7.4. The lysate was ultracentrifuged at
80,000g for 10 minutes, and the supernatant was removed. The
membrane ghosts were washed four times using the phosphate lysing
buffer before being resuspended with 80 mg of unlabeled carrier Hb and made into globin.
Density gradient centrifugation.
The effect of ageing on the hemoglobin composition of circulating red
blood cells was analyzed by density gradient centrifugation. Washed
erythrocytes from an untransfused HbE/+ thalassemic were
centrifuged at 35,000g on a 20% Percoll (Pharmacia, St.
Albans, UK)/meglamine gradient for 20 minutes.19 The red blood cells were divided into three
fractions: a denser older fraction, an intermediate fraction, and a
less dense, younger fraction. These fractions were then washed three
times in reticulocyte saline and the hemoglobin composition of each was
assessed by cation exchange high-performance liquid chromatography
(HPLC). Pyruvate kinase (PK) and glucose-6-phosphate
dehydrogenase (G6PD) levels were also measured in each fraction to
confirm that the age of the cells increased with density.20
| |
RESULTS |
Time-course globin-chain synthesis studies at 37°C.
Time-course studies at 37°C were performed over 2 hours on 5 individuals with HbE/ thalassemia; DNA analysis showed the thalassemia mutations present to be 2 cases of IVS 1-5 (G-C) (severe +), Fr 8/9 (°), IVS 1-130 (°), and IVS
II-654 (°). The steady-state hemoglobin levels ranged from 6.6 to 10.4 g/dL (mean, 8.0 g/dL). Linear synthesis occurred in each case
over the period of the incubation, suggesting that HbE is not
significantly unstable over 2 hours (Fig
1). The mean ratio of (SA increase of E in the first
hour)/(SA increase in E the second hour) (SA 1 hour/SA 2 hours) was 0.94 (range, 0.7 to 1.1;
Fig 2). A ratio of 1 results from linear
synthesis with no globin instability. This result was confirmed in a
sample of bone marrow from an HbE/ thalassemic [IVS 1-5(G-C)] and
using whole, unwashed, blood [IVS 1-5(G-C)], with SA 1 hour/SA
2 hours ratios of 1.1 and 0.92, respectively; the latter experiment was performed to ensure that washing the cells did not remove any small
molecules that might be important in exacerbating HbE instability, eg,
free iron and hydrogen peroxide.21
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| Fig 1.
Specific activity (SA) time-course over 2 hours in an
untransfused patient with HbE/ thalassemia (IVS 1-130). The
incubation temperatures are 37°C (A) and 41°C (B). Synthesis is
linear at 37°C, suggesting that there is no significant hemoglobin
instability. At 41°C, the curve flattens after 1 hour, consistent
with hemoglobin instability. ( )  ; ( ) E; ( )
.
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| Fig 2.
Summary of the results of time-course globin chain
synthesis studies. Results are expressed as the ratio of the increase
in specific activity of the or E chain in the first
hour to the increase in the second hour. The log10 of the
ratio is plotted. If SA increases linearly, the ratio is 1 (log10 0); as the hemoglobin becomes unstable and the SA
increases less in the second hour, the ratio increases exponentially.
() Incubation at 37°C, ( ) incubation at 39°C, ()
incubation at 41°C, ( ) sample was also deficient for pyrimidine
5' nucleotidase. E/beta thal, E/ thalassemia (includes bone marrow
sample and unwashed, whole blood); EE, homozygous HbE; TI, thalassemia
intermedia. The log(SA 1 hour/SA 2 hours) is 0 in HbE/
thalassemia at 37°C, but increases markedly at higher temperatures.
This effect is not seen in thalassemia intermedia.
|
|
Pulse-chase studies at 37°C.
A pulse-chase incubation at 37°C was performed on an HbE/
thalassemic (IVS 1-5 G-C) over 30 minutes to detect any early
hemoglobin instability (Fig 3). The
specific activity increased in the first 5 minutes, as the residual
radioactivity not removed by washing was incorporated; in the following
25 minutes, there was no significant change in the specific activities
of any of the globin chains, suggesting that newly synthesized globin
is not immediately lost.
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| Fig 3.
Pulse-chase globin chain synthesis in an untransfused
HbE/ thalassemic [IVS 1-5(G-C)] at 37°C. At time 0 minutes,
the mixture was washed and the unused radiolabeled leucine was removed.
The SA increases for the first 5 minutes as residual radiolabeled
leucine is used and then plateaus, with no decrease in SA of the globin
chains.() ; ( ) A; () E; ()
.
|
|
Membrane studies at 37°C.
Pulse-chase incubations were performed over 12 hours using blood from
an HbE/° thalassemic (codon 16, -C). At 30 minutes, a
radioactive peak corresponding to newly synthesized globin was
found to be bound to the membrane; no E chain was
observed. At 2 hours, a similar pattern was found with slightly less
radioactive incorporation in the globin peak. By 12 hours, no
radioactive globin was found bound to the membrane (Fig 4). The pattern of
globin binding contrasts to the findings when 60 minutes of incubation
were performed on blood from a known unstable hemoglobin, Hb Bristol
(67 Val-Met
Asp), when both and globin chains were
found on the membrane (Fig 5). This pattern of membrane-bound globin chains was also observed with Hbs Sun Prairie
and Ann Arbor (data not shown).
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| Fig 4.
Cation exchange chromatography of
pulse-radiolabeled globin chains in an HbE/ thalassemic (Cd16) at
37°C, showing globin binding to the membrane 30 minutes, 2 hours,
and 12 hours after removal of the radiolabeled leucine. The arrows show
the position of each globin peak as determined by the separation of the
nonradiolabeled protein. The vertical axis shows counts per minute
(cpm). The initial peak of globin is lost after 12 hours.
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| Fig 5.
Cation exchange chromatography showing globin binding to
the membrane in Hb Bristol (67 Val- MetAsp) after 60 minutes of incubation with tritiated leucine. Binding of both and
globin occurs.
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|
Globin synthesis studies at 41°C.
The 2-hour time-course incubations were repeated at 41°C on the
HbE/ thalassemics with the Fr 8/9 and IVS 1-130 mutations. At the
higher temperature, there is a marked decrease in the rate of increase
of specific activity, typical of hemoglobin instability (Fig 1); this
pattern is similar to that seen with unstable hemoglobins at
37°C.22 Two time-course incubations of HbE/
thalassemia at 39°C also showed a similar plateau. The geometric
mean of SA 1 hour/SA 2 hours at higher temperatures was 25 (range, 2.2 to 792; Fig 2), showing a marked increase compared with
incubations at 37°C; this wide range reflects the fact that, as the
SA increase in the second hour decreases towards zero, the ratio
increases exponentially. This contrasted to a control experiment in
which blood from an untransfused patient with thalassemia intermedia (Cd39/IVS 1-6) showed linear increases in specific activity with time
at both 37°C and 41°C with SA 1 hour/SA 2 hours ratios of
1.2 and 1.3, respectively (Fig 2). Incubations on three controls with
no hemoglobinopathies gave similar results, with SA 1 hour/SA 2 hours ratios around 1. Increased temperature also lead to a decrease in
/non- globin chain synthesis ratios, as shown by the specific
activities and total chain synthesis ratios: after 60 minutes the
/non- ratios were 2.1 and 2.3 in the two HbE/ thalassemia
samples at 37°C and 1.6 and 1.9 at 41°C. Both E
and increased relative to . This temperature effect has been noted before,16 but its explanation and significance are
unclear.
The pattern of hemoglobin binding to the membrane in HbE/
thalassemia at 41°C was similar to that at 37°C, with globin but no E chains bound.
Studies on HbE homozygotes.
Parallel experiments were performed on the red blood cells of a HbE
homozygote. Time-course studies at 37°C showed linear synthesis as
expected, but increasing the temperature to 41°C showed the
development of a plateau, similar to that in HbE/ thalassemia. The
SA 1 hour/SA 2 hours ratio increased from 0.7 at 37°C to 18 at 41°C. In contrast to HbE/ thalassemia, membrane analysis
showed little globin binding at 37°C and 41°C, although at the
higher temperature there was a small peak in the E
position.
Density gradient centrifugation.
Density-gradient-separated fractions of blood from an
HbE/+ thalassemic [IVS 1-5(G-C)] were analyzed for
hemoglobin composition, with the centrifugation and chromatography
being performed in duplicate. The results are summarized in
Fig 6. There is a relative increase in HbF,
mirrored by a decrease in Hbs E and A in the older, denser fraction.
The HbF/HbA ratio increased from 5.3 in the younger fraction to 9.2 in
the older. On the other hand, there was no significant
change in the HbE/HbA ratio across the gradient, indicating that there
was no loss of the HbE relative to HbA during the lifespan of red blood
cells. The pyruvate kinase activity decreased from 9.6 IU/1010 red blood cells in the top fraction to 4.15 in the
bottom; G6PD levels fell similarly from 3.71 IU to 2.44.
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| Fig 6.
Hb composition of three fractions of erythrocytes
separated by density gradient centrifugation of blood from an
untransfused HbE/+ thalassemic [IVS 1-5(G-C)],
separating red blood cells into top (least dense, youngest cells),
middle, and bottom (densest, oldest cells). Each point is the average
of two centrifugations from the same patient. The ratio of HbE/HbA
increases slightly as the red blood cells age, suggesting that HbE is
at least as stable as HbA; the ratio of HbF/HbA increases with cell age
as expected. The decrease in pyruvate kinase activity with increasing
cell density confirms that the denser fractions contain predominantly
older red blood cells. () %E/%A; () %F/%A; (×) PK.
|
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| |
DISCUSSION |
This is the first study to address directly the question of HbE
instability in its most important clinical context, HbE/ thalassemia. There is little previously published evidence either for
or against the clinical importance of the instability of HbE in vivo.
There is a single case report of dapsone causing a Heinz body hemolytic
anemia in a Cambodian woman with HbE trait,23 although this
has not been confirmed by subsequent observations, despite the
widespread use of the drug in areas where HbE is prevalent. A second
study indirectly implicated HbE instability as important in protection
against malaria, by noting that HbE only offered protection if fava
beans had been consumed in the preceding month.24 We have
shown previously that pyrimidine 5' nucleotidase deficiency interacts
with HbE to produce hemoglobin instability and hemolysis.11 However, there are a large number of reports of HbE occurring with
other hemoglobin and red blood cell defects, without any suggestion of
interaction: these include HbE with HbS,25
HbC,26 Hb Lepore,27 Hb Hope,28 G6PD
deficiency,29-31 and hereditary elliptocytosis.32,33 The interaction between HbE and HbH
disease is particularly interesting: the resulting condition,
HbAE-Bart's disease, is similar in severity to HbH
disease,28 suggesting that the HbE does not ameliorate the
condition by reducing globin chain imbalance or exacerbate it by
maximizing the hemolytic component.
The pattern of globin binding to the red blood cell membrane at
37°C is similar to that found in thalassemia major,18
with only globin present, and has been confirmed by electron
microscope immunocytochemical studies of inclusions in HbE/
thalassemia.34 Time-course studies show that newly bound
globin is proteolysed within 2 and 12 hours of precipitation. This
suggests that the amount of globin bound to the membrane in thalassemia is determined by a dynamic equilibrium between the rate of
precipitation and the rate of hemolysis and indicates that the majority
of globin-induced damage to the membrane may occur late in development
of the red blood cell, because the proteolytic capacity of the
reticulocyte is very limited compared with that of the earlier
nucleated red blood cell precursors.35,36
We have found no evidence that instability of HbE is important in the
steady state in HbE/ thalassemia. Time- course globin chain
synthesis experiments showed linear synthesis at 37°C in both
marrow and peripheral blood (Fig 2). Pulse-chase studies showed that
only globin, not E globin, binds to the red blood
cell membrane, in contrast to other unstable hemoglobins such as Hb
Bristol.
Density gradient centrifugation of red blood cells from a
HbE/+ thalassemic supported these findings (Fig 6).
There is good evidence that in most conditions red blood cells become
more dense as they age; sickle cell syndromes appear to be the
exception to this.37 In our experiment, the decreasing
levels of PK and G6PD activity associated with increasing density
strongly suggest that the denser fractions are predominantly made up of
older cells. The relative increase in HbF with cell age reflects the
well-defined survival advantage of cells containing more
HbF,38 but there was no significant loss of HbE relative to
HbA in the older cell population. Taken together, these data suggest
that HbE is not significantly unstable in vivo in the steady state.
However, the globin chain synthesis studies at higher temperatures
suggest that newly synthesized hemoglobin is unstable in HbE/
thalassemia, in contrast to the effect of increasing temperature on
control cells from a patient with thalassemia intermedia. The
instability involved , E, and chains, suggesting
that hemoglobin tetramers containing E chains are
precipitated, rather than free globin chains, a situation that has been
observed previously with unstable hemoglobins.22 Instability occurred at both 39°C and 41°C, both significantly above body temperature; however, in tropical regions where malaria is
endemic, febrile illnesses are common26 and the resulting exacerbation of dyserythropoiesis and hemolysis might be a significant cause of variability in hemoglobin levels in cross-sectional studies. It is known that fevers cause increased hemolysis in patients with
unstable hemoglobins39 and hemoglobin H
disease.40 There is an anecdotal report that fevers cause a
more marked decrease in hemoglobin in HbE/ thalassemia and HbE
disease,41 but this potentially important observation has
not been studied subsequently.
Are there other possible interpretations of the result of the globin
chain synthesis studies at higher temperatures? One possible explanation for the plateau in the rate of increase of specific activity is that the rate of mRNA translation starts to decrease over 2 hours at 41°C; the maintenance of linear synthesis in thalassemia intermedia at 41°C goes against this. Although the hemoglobin appears to be unstable at higher temperatures in HbE/ thalassemia, the pattern of globin binding to the membrane is unchanged compared with that at 37°C, with only globin found. This suggests that precipitated E chains are either proteolysed rapidly
from the membrane or that they are unable to bind to the membrane at
all. This absence of globin binding to the membrane is similar to
that in the interaction between HbE and pyrimidine 5 nucleotidase
deficiency.11 It is also interesting that the HbE
homozygote showed a similar temperature effect to HbE/ thalassemics,
with a plateau occurring in specific activity after 1 hour at 41°C
and an SA 1 hour/SA 2 hours ratio of 18; it differed, however, in
that no definite detectable peak of radioactivity was found attached to
the membrane. The clinical correlate of this finding in homozygous HbE
is unclear, with little information on the effects of fever on
homozygotes.
This study suggests that the nature of the interaction between HbE and
thalassemia is primarily the interaction of two thalassemia
alleles and that the resulting degree of anemia is related to the
amount of globin chain imbalance. Similarly, the marked variability in
severity between different individuals with HbE/ thalassemia
principally results from differences in globin chain imbalance,
including variability in chain synthesis and coexistent thalassemia. However, we have also found evidence that HbE can be
significantly unstable when subjected to mild oxidative stress, such as
increases in body temperature, and this may also contribute to the
variability and severity. The significance of these temperature effects
needs to be addressed in clinical studies.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted May 12, 1998.
Supported by the Medical Research Council, UK.
Address reprint requests to D.C. Rees, MRCP, MRC Molecular
Haematology Unit, University of Oxford, Institute of Molecular Medicine, The John Radcliffe, Headley Way, Headington, Oxford, OX3 9DS, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr J.B. Porter, Dr C. Hatton, Dr P. Darbyshire, and
Dr A. Yardumian for referring patients used in this study and J. Darley
for red blood cell enzyme assays.
| |
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Abstract
Introduction
Abstract
