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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4375-4382
HbS-Oman Heterozygote: A New Dominant Sickle Syndrome
By
Ronald L. Nagel,
Shahina Daar,
Jose R. Romero,
Sandra M. Suzuka,
David Gravell,
Eric Bouhassira,
Robert S. Schwartz,
Mary E. Fabry, and
Rajagopal Krishnamoorthy
From the Division of Hematology, Albert Einstein College of
Medicine/Montefiore Medical Center, Bronx, NY; the Division of
Endocrinology and Hypertension, Brigham and Women's Hospital, Harvard
Medical School, Boston, MA; Hôpital Robert Debré, Paris,
France; and the Department of Hematology, Hospital Sultan Qaboos
Medical School, Muscat, Sultanate of Oman.
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ABSTRACT |
Hemoglobin (Hb) S-Oman has two mutations in the  -chains. In
addition to the classic S mutation (6 Glu  Val), it contains a second mutation in the same chain (121 Glu
Lys) identical to that of HbOARAB. We have studied a pedigree of heterozygous carriers of HbS-Oman that segregates into two types of patients: those expressing about 20% HbS-Oman and
concomitant   / thalassemia and those with about 14% of HbS-Oman and concomitant / thalassemia. The higher
expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome
of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in
Oman. In addition, the higher expressors exhibit a unique form of
irreversibly sickled cell reminiscent of a "yarn and knitting needle" shape, in addition to folded and target cells. The
CSAT of S-Oman is identical to that of S-Antilles, another
supersickling hemoglobin, whose carriers express the abnormal
hemoglobin at 40% to 50%, with a very similar clinical picture to
HbS-Oman. Because the level of expression is so different and the
clinical picture so similar, and based on the hemolysates
CSAT's, we conclude that HbS-Oman produces pathology
beyond its sickling tendencies. A clue for this additional pathogenesis
is found in the fact that homozygous HbOARAB, which has the
same second substitution as S-Oman, has a moderately severe hemolytic
anemia; when HbOARAB is combined with HbS, it makes the
phenotype of this double heterozygote as severe as SS. Properties of
HbS-Oman red blood cells (RBCs) include reticulocytes that are much
denser than normal (similar to those of SC and CC
disease), a decrease in the Km for
Ca2+ needed to activate the Gardos' channel (making this
transporter more sensitive to Ca2+), increased
association of HbS-Oman with the RBC membrane, the presence of dense
cells by isopycnic gradient, the presence of folded cells, and abundant
nidus of polymerization under the membrane. Other properties
include a clear increase in volume and
N-ethylmaleimide-stimulated K:Cl cotransport in RBCs
expressing more than 20% HbS-Oman. We conclude that the pathology of
heterozygous S-Oman is the product of the sickling properties of the
6 Val mutation which are enhanced by the second mutation at 121.
In addition, the syndrome is further enhanced by a hemolytic anemia
induced by the mutation at 121. We speculate that this pathology
results from the abnormal association of the highly positively charged
HbS-Oman (3 charges different from normal hemoglobin) with the RBC
membrane.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
SICKLE TRAIT red blood cells (RBCs)
usually do not sickle at pH 7.4 and the peripheral blood of carriers
does not contain dense cells, irreversibly sickled cells, or exhibit
increased reticulocyte counts. Furthermore, sickle trait individuals do not have splenomegaly. Hence, it is of particular interest that RBCs
containing about 20% (the balance being hemoglobin [Hb] A) of a
super polymerizing HbS, which is the result of two mutations (S-Oman = 6 Glu Val; 121 Glu Lys) in the -chain, have been observed to contain bizarrely deformed
sickled cells (ISCs) in peripheral blood.1
The increased sickling tendencies of HbS-Oman is the consequence of the
pro-sickling effects of the 121 mutation, Glu Lys, which
is also found as a single mutation in HbOARAB. The latter hemoglobin, when combined with HbS, produces a syndrome more severe than SC, but easily confused with SC because of the same
electrophoretic pattern on cellulose acetate.
Purified S-Oman has a CSAT (solubility of the deoxy
polymer) of 11 g/dL, much lower than HbS alone (CSAT = 17.8 g/dL).1 This result is interesting because another double
mutant (Hb S-Antilles = 6 Glu Val; 23 Val Ile), has a similarly low CSAT and much higher expression
(40% to 50%) in the trait form, but has a phenotype that is similar
in intensity to the trait form of HbS-Oman.2 Hence, the
identical intrinsic tendency to polymerize of these two super HbSs is
in contradiction with the phenotype: S-Oman has the same effect as
S-Antilles, but with half the level of expression. We need to look for
other pathogenic properties of S-Oman that must be cellular in
character.
In this report we examine the phenotype of S-Oman trait, in a larger
number of individuals, and in more detail than in the original
description of this abnormal Hb.1 We have also studied the
interaction of this abnormal Hb with the RBC membrane and explored its
capacity to increase the density of reticulocytes and to produce dense
cells.
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MATERIALS AND METHODS |
Hematological parameters and cellular studies.
Blood counts (RBC, white blood cell [WBC], platelet count, mean cell
volume [MCV], mean cell hemoglobin concentration [MCHC], and mean
cell hemoglobin [MCH]) were performed with a Technicon 1 (Tarrytown, NY). HbF levels were determined by alkaline denaturation and agar electrophoresis with the standard methods. Deoxygenation, SEM,
and TEM were performed also as described
elsewhere.3
Hemolysate CSAT.
CSAT values were determined in a 0.1 mol/L potassium
phosphate buffer, pH 7.35, at 25°C. The hemolysate was first
deoxygenated with alternate vacuum and N2 and then Na
dithionite was added to three times the final concentration of Hb.
Samples were allowed to gel overnight at 25°C and were centrifuged
the next day at 25°C for 2 hours at 35,000 rpm. Purified HbS in the
same buffer was run as a control. The supernatants were removed
anaerobically and Hb concentrations and deoxy pHs were determined.
Isoelectric focusing and high-performance liquid chromatography
(HPLC).
The methods involved were conducted as described
elsewhere.3
RBC membrane association with HbS-Oman and HbA in S-Oman
heterozygote RBCs.
RBCs were lysed with distilled water and the sample was centrifuged to
obtain supernatant 1, following the technique of Klipstein and
Ranney.4 The RBC membranes obtained after removing
supernatant 1 were extracted with an 8.6 pH buffer solution. The
supernatant obtained after centrifugation was labeled supernatant 2. Both supernatants where analyzed by isoelectric focusing and HPLC.
HbS-Oman binding to normal RBC membranes.
"Leaky" RBC membranes (ghosts) were prepared from a
hematologically normal (HbAA) individual as follows: RBCs were pelleted from whole blood by centrifugation at 500g for 10 minutes at
4°C, and washed three times with 5 mmol/L sodium phosphate, pH 7.4, 140 mmol/L sodium chloride (PBS). Each time the small buffy coat was
carefully discarded. The washed RBCs were lysed with 5 mmol/L sodium
phosphate, pH 8.0 (5P8) and the RBC membranes (ghosts) were collected
by centrifugation at 15,000g for 15 minutes at 4°C. The
membranes were washed with 5P8 until white. Residual Hb and glycolytic
enzymes were stripped by incubating the ghosts with 20 vol of 10 mmol/L
phosphate, pH 7.5, 0.5 mol/L sodium chloride for 30 minutes on ice. The
ghosts were collected by centrifugation, as described above, and washed
once with 10 mmol/L sodium phosphate pH 6.0 (10P6). Under these
conditions, the ghosts do not reseal and are thus "leaky." An
aliquot was taken for protein determination using the BCA
method (Pierce Chemical Co, Rockford, IL) after solubilization in 1%
sodium dodecyl sulfate (SDS).
The ghosts (1 mg/mL final concentration) were incubated with purified
HbAA or HbS-Oman (20 mg/mL final concentration) in 10P6 (final
concentration) for 90 minutes at 4°C in a total volume of 0.2 mL
while rotating. The incubations were run in triplicate. After the
incubation, ghosts were collected by centrifugation, as described
above, and washed vigorously four times with ice-cold 10P6. After the
final wash, the ghosts were resuspended in 0.2 mL ice-cold 10P6 and
0.04 mL was removed for SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) analysis (see below). The remainder was solubilized by
addition of 2 vol of 20 mmol/L sodium phosphate, pH 7.4, 125 mmol/L
sodium chloride, 1.5% Triton X-100. Hb in the solubilized ghosts was
measured using Drabkin's reagent (Sigma, St Louis, MO), and compared
with an Hb standard. Hb binding results were expressed as moles of Hb
dimer bound per mole of ghost band 3. It was assumed that band 3 represents 30% of ghost total protein and has a molecular weight
(MWr) of 90 kD.
SDS-PAGE.
Ghosts, 0.02 mg, were solubilized and the proteins separated by
SDS-PAGE on 7.5% acrylamide gels using the Fairbanks continuous buffer
system. The gels were stained with Coomassie brilliant blue, and the
protein bands were quantitated by scanning laser densitometry.
Gradients (analytical and preparative).
RBCs were either used for determination of density distribution or
separated by density gradient centrifugation into three fractions on a
Percoll (colloidal silica coated with polyvinyl pyrrolidone; Pharmacia
Fine Chemicals, Inc, Piscataway, NJ) and Larex (arabinogalactan
polysaccharide; Larex International Inc, Roseville, MN) gradient as
previously described.5,6
Measurements of RBC Ca2+-activated K+
efflux.
The maximal activity of the Ca2+-activated K+
efflux was determined under optimized conditions in the presence of the
calcium-ionophore A23187 as previously described by Romero et
al7 in the presence and absence of 10 µmol/L clotrimazole
(CLT). CLT has been shown to be a potent, high-affinity inhibitor of
Ca2+-activated K+ channels (Gardos Channel) in
human RBCs.8 The ionophore A23187 was used to increase
cytosolic ionized Ca2+ and clamp it at various levels. Net
K+ efflux was determined by incubating cells in an
Na+ media containing the A23187 (60 µmol/L of RBCs). The
Na+ media contained (mmol/L): 140 NaCl, 10 Tris-MOPS pH 7.4 at 37°C, 0.1 ouabain (to inhibit the Na+ pump), 10 glucose, 0.15 MgCl2, 0.1 bumetanide (to inhibit
Na+:K+:2Cl cotransport), 1 EGTA or 1 Tris-citrate; and total CaCl2 varied between 0 and 100 µmol/L, A23187 (60 µmol/L of RBCs), at 1%
hematocrit. This gives a cytosolic ionized
Ca2+ that is between <0.1 nmol/L Ca2+ with 1 mmol/L EGTA and 20 µmol/L Ca2+ with 1 mmol/L citrate.
Cytosolic ionized Ca2+ was buffered at <0.1 nmol/L
Ca2+ with 1 mmol/L EGTA or at  5 µmol/L Ca2+
with 1 mmol/L citrate. Initial rates of K+ efflux in RBCs
were measured by sampling the efflux media in duplicate at 1, 3, 5, and
7 minutes in the presence or absence of 10 µmol/L CLT. In experiments
where the Ca2+ was reduced to <0.1 nmol/L, the samples
were taken at 1, 3.5, and 7 minutes. K+ efflux was
calculated from the slope of the regression line of K+
concentration versus time taking into account the volume of RBCs used.
The slope ± SE of the linear regression was calculated using Enzfitter software for the personal computer. The
CLT-sensitive Ca2+-activated K+ efflux was
estimated by subtracting the flux in the presence of CLT from the total
flux at various cytosolic calciums. The CLT-sensitive K+
efflux was then analyzed as a function of the cytosolic ionized calcium
using a nonlinear regression analysis. This allowed for estimation of
the Vmax and Km for calcium stimulation of
Ca2+-activated K+ efflux.
The total calcium concentration of the flux media was measured by
atomic absorption spectrophotometry using calcium standards (EM
Sciences, Cherry Hill, NJ) in sodium media. A custom-made computer
program was used to calculate the Ca2+ concentrations using
the dissociation constant and correcting for ionic strength at pH 7.4 and 0.15 mmol/L MgCl2 as previously described.8
CLT was obtained from Sigma (St Louis, MO).
Measurements of volume and N-ethylmaleimide
(NEM)-stimulated K+ fluxes.
The protocol followed was identical to the ones described in Romero et
al.7
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RESULTS |
Clinical and laboratory values in five carriers of HbS-Oman.
The sample consists of three individuals with 20% to 23% Hb-S Oman
(HSR, MSR, ISH) and two with levels of 13% and 14% (SSR, MSK)
(Table 1 and pedigree in
Fig 1). In the high S-Oman group all
have silent carrier status for -thalassemia, and correspondingly, they have moderately reduced MCV and MCH (70 to 76 fL and 23 to 25 pg).
The second group, with low HbS-Oman expression, have concomitantly / status, and hence significantly lower MCV and MCH
(66 to 67 fL and 68 fL and 21.4 to 21.7 pg). The level of anemia was also different between these groups: individuals with higher S-Oman had
lower Hb than those with low levels of S-Oman and tended to have
enlarged spleens. Correspondingly, reticulocyte counts tend to be lower
in the latter than in the former. Nevertheless, the patient ISH is an
exception, because she had a low reticulocyte count and the spleen was
not palpable, probably due to young age. Finally, the HbF levels are
only mildly to slightly elevated.
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| Fig 1.
Pedigree of the five heterozygous carriers of HbS-Oman
described in Table 1. Hatched symbols indicate the presence of HbS-Oman
in the heterozygous state. The question marks indicate the obligatory
heterozygotes according to the pedigree.
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Clinical records of three patients with high S-Oman expression show the
following: MSR, 11 years old, had frequent painful crises and episodes
of hypoxic encephalopathy and acute chest syndrome, qualifying for a
severe phenotype; ISH, 6 years old, has frequent but mild painful
crises; a third patient, HSR, 18 years old, with 22.3% S-Oman,
required frequent transfusions between the ages of 3 and 10 and has had
mild painful crises afterward. MoSR, a patient not included in the
present series, 23 years old, 27% S-Oman, has had acute painful
crises, acute chest syndrome, and episodes of pain in the shoulders.
The two patients with 14% S-Oman (Table 1), plus another patient,
female, 45 years old and 13.3% S-Oman (ZSK), report no clinical
symptomatology.
Isopycnic gradient distribution of RBCs in S-Oman carriers.
Figure
2 depicts the isopycnic gradients for the five carriers. All of the
patients had more light cells at the top of gradient, a consequence of
the presence of -thalassemia and hemolysis. The percent dense cells,
compared with an AA subject, were increased in two of the
high S-Oman expressors and less in the other three individuals. Not
surprisingly, the exception was ISH, who was also different because
she did not have a high reticulocyte count or a palpable
spleen, and was only 6 years old. Dense cells are greatly decreased in
early childhood sickle syndromes, most likely due to the presence of a
relatively efficient spleen.
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| Fig 2.
Percoll-Larex gradients of the five members of the
pedigree. Lane B, density beads; lane 1, normal blood; lane
2, sickle cell anemia; lane 3, HSR (high HbS-Oman); lane
4, ISH (high HbS-Oman); lane 5 MSR (high HbS-Oman); lane 6 MSK (low
HbS-Oman); lane 7 SSR (low HbS-Oman).
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The isopycnic gradients were also used preparatively, which allowed
separation of the cells into fractions 1, 2, and 3 of increasing
density, which were then analyzed for the percent reticulocytes and
irreversibly sickled cells. Figure 3
depicts the percent reticulocytes as a function of increasing RBC
density in both a low S-Oman patient (SSR; 14% S-Oman) and a high
S-Oman patient (MSR; 20% S-Oman). Interestingly, the highest
percentage in SSR is in the lightest fraction (F1), while in the high
S-Oman patient, reticulocytes were located preferentially in the
densest fraction (F3).
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| Fig 3.
Reticulocyte count in three increasingly dense fractions
isolated from the density gradient. The high S-Oman patient MSR ( )
and the low S-Oman patient SSR ( ). Notice that the highest percent
of reticulocytes is found in F1 of SSR as is observed for AA, AS, and
SS patients. The highest percent reticulocytes is found in F3 (the
densest) in MSR, as is observed in CC and SC patients.
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Blood smear and sickling of S-Oman trait RBCs.
The most striking feature of the smear of the higher expressors of
HbS-Oman was the presence of cells which appeared to be the consequence
of a single polymerized domain of HbS-Oman traversing the RBC. Although
the level of dehydration of these cells varied, they most frequently
had little or no dehydration (Figs 4 [see page 4378] and
5). They suggest a "yarn/knitting
needle" shape. These forms were very rare or absent in the blood of
S-Oman patients with less than 15% S-Oman. These cells, when
oxygenated, are devoid of polymer but retain their shape, qualifying
for the name irreversibly sickled cells. When deoxygenated,
they formed single or double domains, suggesting the presence of single
or double nucleation events (Fig 6). Near
the membrane, bunches of polymer were observed (Fig 6).
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| Fig 4.
Hematoxyline-eosine stained smear of a patient with high
S-Oman expression. The characteristic shapes that resemble a ball of
yarn transversed by knitting needles of the irreversibly sickled cells
found in these patients is clearly visible. Also visible are target
cells and other deformed cells, including folded cells.
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| Fig 5.
Scanning electron microscopy of RBCs from a high S-Oman
patient. (A) Oxygenated; (B) deoxygenated. Notice the yarn/knitting
needle shape of the irreversibly sickled cells characteristic of this
disease, highlighted by white arrows. Notice that after full
deoxygenation almost all of the cells sickled with long, thin
protuberances. The lower two panels (C) and (D) are of a low S-Oman
patient. (C) Oxygenated, looks quite normal except for a few
echinocytic and otherwise deformed RBCs. No yarn/knitting needle cells.
(D) Fully deoxygenated cells; again, almost all of the cells sickle,
but with less elongated and numerous protuberances.
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| Fig 6.
Transmission electron microscopy of "yarn/knitting
needle" RBCs when deoxygenated in vitro. "Yarn/knitting" RBCs
are irreversibly sickled cells, that is their shape does not change
according to the presence or absence of intracellular polymer. These
cells were likely formed by the presence of a single domain of polymer
transecting the cell and growing to the extent that the membrane loses
its elasticity and the deformation becomes permanent. By deoxygenating
peripheral blood from a patient with a high expression of S-Oman we
observed the type of domain formation that occurs in these cells.
Notice that a single large domain is forming in the center (B and C),
but much smaller domains are also formed, in the distal extension of
the membrane as well as immediately under the membrane, as magnified in
(D). (A) This RBC has a "banana" shape, capable of accommodating
two long polymer domains separated by an angle approaching 180°:
indeed, two major domains are being formed upon in vitro deoxygenation
in these cells, following the requirement of the already established
irreversibly deformed shape.
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CSAT results.
Table 2 reports on the CSAT of
hemolysates of SSR, a low HbS-Oman expressor, and ISH, a high S-Oman
expressor, which are significantly different. The patient SSR, who is
asymptomatic and has 14% S-Oman, has a CSAT significantly
higher than sickle trait (an increase of  4 g/dL) that is similar to
50% mixtures of HbF and HbS, a fact that explains the lack of a
pathological phenotype. The results on ISH are particularly
interesting: the CSAT is very close to that of sickle
trait. In other words, the phenotype is more severe than the
CSAT predicts. Hence, we have to search for the
nonpolymerization-dependent effect of HbS-Oman to explain the
phenotype.
Membrane retention of Hb by the RBC membrane after distilled water
RBC lysis.
Hb retention was studied by comparing the Hb composition of
supernatants 1 and 2 (Fig 7). Supernatant
1, generated by distilled water lysis, had percents of HbA and
HbS-Oman, similar in proportion to the regular hemolysate. Supernatant
2, obtained by extracting the Hb left in the RBC ghosts after distilled
water lysis, was enriched in HbS-Oman. For the patient with 15% S-Oman
in the regular hemolysate, quantification of the bands in supernantant
2 (representing the Hb attached to the membrane after distilled water
lysis) (Fig 5) gave a percent of HbS-Oman of 73.2% and HbA of 17.3%
compared with 14.1% S-Oman and 57% HbA in supernatant 1. The other
sample, from a carrier with 20% HbS-Oman in the regular hemolysate,
had a very similar distribution, with S-Oman 69.4% and HbA 20.7%
compared, numbers that are quite different from the distribution in
supernatant 1 (20.8% S-Oman and 58.4% HbA). These numbers do not add
up to 100% because IEF separates also Hbs A1c,
S-OmanIc, F, and A2, not relevant to this
analysis.
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| Fig 7.
Isoelectrofocusing separation of the Hb tetramers present
in supernatant 1 and supernatant 2 (see Materials and Methods). Lane 1, markers for HbS A, F, S, and C(A2) (from the top down).
Lane 2, supernatant 1 of a normal hemolysate (HbA, traces of HbF and
low levels of HbA2; the bands in front of HbA are
postranslational modifications of HbA). Lane 3, supernatant 1 of
patient SSR (HbA, low amounts of HbF and HbA2 and the
slowest fraction, HbS-Oman). Lane 4, supernatant 1 of patient ISH (high
S-Oman, similar distribution to that in 3 but less loaded). Lane 5, supernatant 2 of a normal hemolysate (HbA and very little
A2). Lane 6, supernatant 2 of patient SSR, dramatic
reduction of HbA and large increase in HbS-Oman. Lane 7, supernatant of
patient ISH, very similar results that in lane 6. For quantification,
see the text.
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Binding of HbS-Oman to RBC "leaky" ghosts.
In two separate experiments we compared the binding of HbS-Oman and HbA
to RBC ghosts. The results of one of these experiments are depicted in
Table 3. The second experiment gave the
same results: there is a threefold increase in binding of HbS-Oman to
"leaky" ghost compared with HbA. In addition, gel electrophoresis confirmed the significantly increased binding of HbS-Oman to these ghosts (Fig 8 [see this page]).
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| Fig 8.
Increased binding of HbS-Oman to normal (HbAA)
Hb-stripped "leaky" RBC ghost membranes. SDS-PAGE (7.5%
acrylamide), using the Fairbanks discontinuous buffer system, stained
with Coomassie brilliant blue of purified HbAA or HbS-Oman incubated
with normal (HbAA) Hb-stripped "leaky" RBC ghost
membranes. Lane 1, normal (HbAA) Hb-stripped
"leaky" ghosts; lane 2, 0.005 mg purified HbS-Oman; lanes 3 through 5, "leaky" ghosts incubated with HbAA; lanes 6 and 10, "leaky" ghosts without added Hb; lanes 7 through 9, "leaky"
ghosts incubated with HbS-Oman. The position of band 3 and Hb is
indicated. There was considerably more binding of HbS-Oman to
"leaky" ghosts compared with the binding of HbAA.
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Ca2+-activated K+ efflux.
Human RBCs possess a Ca2+-activated, charybdotoxin (CTX)
sensitive K+ efflux8-10 that is also sensitive
to CLT.8 Experiments on RBCs from an SS patient showed that
the presence of 20 µmol/L cytosolic Ca2+ and A23187
induced a maximal stimulation of the K+ efflux (4.27 ± 0.57 mmol/L cell × min). This efflux was reduced to 0.16 ± 0.19 mmol/L cell × min when the RBCs were incubated with 10 µmol/L CLT in the presence of 20 µmol/L cytosolic Ca2+
and A23187. When cytosolic Ca2+ was buffered at <0.1
nmol/L with EGTA, in the presence of A23187, the K+ efflux
was significantly reduced to 0.07 ± 0.14 mmol/L cell × min.
The difference between K+ efflux in the presence and
absence of CLT yields the  -CLT sensitive fraction, which in this
experiment was 4.11 mmol/L cell × min. Therefore, the percent
inhibition of the total maximal activity of the
Ca2+-activated K+ efflux was approximately 99%
for this experiment.
The kinetic parameters of the CLT-sensitive Ca2+-activated
K+ efflux measurements in RBCs from SS and heterozygous
S-Oman patients is of interest. In both cell types, the
Ca2+-activated K+ efflux was already maximally
stimulated by approximately 10 µmol/L cytosolic calcium. In the
presence of CLT, Ca2+-activated K+ efflux
values were not significantly different in both cell types. Therefore,
the CTX-sensitive K+ efflux was not significantly
different in these cells. However, the Km for stimulation
of the Ca2+-activated K+ efflux was
significantly lower in S-Oman than in SS RBCs. The RBCs from HSR
(S-Oman 22%) had a Km of 0.487 µm and a Vmax
of 6.22 FU while the RBCs of MSK (S-Oman 13%) had a Km of
1.81 µm and Vmax of 6.05 FU. This is in clear contrast to
the values of Km 7.53 µm and 5.5 FU obtained for SS
blood.
Volume- and NEM-stimulated K:Cl cotransport K+.
Three patients were studied: two with less than 14% S-Oman (RHK, SSR)
and one with 20% HbS-Oman (MSR). The volume-stimulated K:Cl
cotransports were 5.6 and 1.2 mmol/L cell × h, respectively, and
the NEM-stimulated K:Cl cotransports were 25.6 and 24.0 mmol/L cell × h in the low S-Oman RBCs. The high S-Oman RBCs exhibited a
volume stimulated K:Cl efflux of 14.7 mmol/L cell × h and an NEM-stimulated K:Cl cotransport of 56.7 mmol/L cell × h. For
comparison, the average values for the Cl-dependent part of volume
stimulated K:Cl cotransport for AA, SS, and CC cells are 2.5 ± 0.5, 15.8 ±1.5, 17.3 ± 1.5 mmol/L cells × h,
respectively. The average value for NEM stimulation for SS RBCs has
greater variability (32.7 ± 10.6 mmol/L cells × h).
| |
DISCUSSION |
The pedigree presented here corresponds to a new sickle syndrome
characterized by the presence of anemia, high reticulocyte count, the
presence of "yarn/knitting-needle" shaped RBCs, splenomegaly, painful crises, and, in one case, central nervous system (CNS) symptoms
and acute chest syndrome. Because all the symptomatic individuals
studied were concomitantly heterozygous for the African-type -thalassemia (/), due to the extremely high
incidence of -thalassemia in Oman,11 we suspect that we
are describing a milder form of this syndrome than would be the case in
the absence of -thalassemia. The clinical syndrome is only observed
in individuals with hemolysates having more than 20% HbS-Oman.
Individuals with a lower percent of the doubly mutated HbS (around
14%) and concomitant homozygosity for the African-type -thalassemia
(/) are asymptomatic clinically and hematologically
have only slight to moderate RBC changes.
The failure of the original investigators who first described
HbS-Oman1 to recognize the clinical syndrome stems from the fact that they had access to a single carrier who happened to have a
concomitant / haplotype and, hence, a low
expression (14%) of S-Oman.
Several factors contribute to the pathogenic effect of this double
mutation. It is clear that the second mutation (121 Glu Lys) has an enhancing effect on polymerization, as shown by the
CSAT of the hemolysate (not S-Oman purified) of a
symptomatic patient, which is the same as that of a sickle trait (AS)
despite the relatively low expression of 22% for S-Oman compared with 40% HbS in sickle trait. S-Oman shows that the effect of 121 Glu
Lys is also apparent in cis (that is, cis to
the active 6Val site).
Nevertheless, the pro-polymerizing effects of 121 (Glu Val) cannot entirely explain the phenotype. Pure S-Oman has a
CSAT of 11 g/dL, which is almost identical to the 11.8 g/dL
CSAT of S-Antilles, another super-hemoglobin S that is the
product of a double mutation in the -chain. The moderate sickle cell
anemia phenotype of S-Antilles trait is the consequence of the
enhancing effect of the 23 Val Ile mutation on
polymerization and a yet undefined effect of the right shift of the
oxygen equilibrium curve observed both in HbS-Antilles as well as the
recombinant hemoglobin 2223Val
 Ile.2,12 Considering the differences in
expression between the two, of almost 40% to 50% for S-Antilles
compared with the 20% to 22% of S-Oman, the phenotype of the latter
should be quite mild or nonexistent. In fact, the phenotypes are very
similar. Finally, the hemolysate CSAT's lend further
support to this analysis. These features suggest that there is an
additional pathogenic effect due to the presence of the 121 (Glu
Lys) mutation.
We propose here that the 121 mutation, by increasing the charge
difference by three, produces an abnormal interaction of the HbS-Oman
tetramers containing the doubly mutated -chain with the RBC
membrane. A strong independent confirmation of this phenomena is the
presence of moderate hemolytic anemia in the homozygotes of
HbOARAB.12,13 Abnormal
interaction with the membrane has been described for HbC (a two-charge
difference)4,4,15 and for HbA2 (a two-charge
difference).4,14 Evidence for an abnormal interaction of
S-Oman with the membrane can be summarized as follows:
The presence of "yarn/knitting needle" cells, which is
characteristic of the syndrome described here and most likely the
consequence of the polymerization of S-Oman in a single domain, is
limited to patients expressing HbS-Oman at the 20% to 22%
level. Electron microscopy of in vitro deoxygenated
S-Oman RBCs shows that cells already deformed to the "yarn/knitting
needle" shape, due to previous events of polymerization that
surpassed the yield coefficient of the membrane, exhibit one or two
domains of polymerization. This represents a low number of nucleation
events, which is not surprising considering the relatively low
concentration of HbS-Oman.
We conclude that the pathophysiology of the sickle syndrome S-Oman
heterozygote is the result of the following gene-product effects:
(1) The sickle mutation (6 Val) induces sickling; (2) The 121 Lys
mutation enhances sickling; and (3) the 121 Lys mutation induces
hemolysis and RBC shape changes.
We know that Hbs with increased positive charge (HbC,
HbOARAB) are by themselves15-17 capable of
reducing the volume of the RBC (with corresponding increase in MCHC),
reducing the RBC life span, and producing morphological changes (folded
cells). In the homozygous form, both of these Hbs produce hemolytic
anemias.16,17 S-Oman heterozygotes have folded cells and
reduced RBC life span, very much like CC and SC cells, although they do
not have the uniformly increased density18 of these
genotypes.
Finally, there is the matter at the low percentages of S-Oman found in
the heterozygotes describe here. The difference in percentage of S-Oman
are associated with the degree of -thalassemia and the numbers are
still low in absolute terms, suggesting that other factors are
involved. Message instability or transcription abnormalities might be
present and need to be excluded. Nevertheless, another interesting
possibility is that the assembly of HbS-Oman is decreased by the very
high positive charge of the S-Oman, as
Bunn19 has suggested for other positively charged Hbs
(C,E,OARAB).
We postulate that the pathogenic mechanisms mediated by S-Oman, in
addition to the facilitation of Hb polymerization, include a direct or
indirect charge-dependent interaction between S-Oman and the RBC
membrane, resulting in changes of the intracellular Hb concentration or
increased cell density, by altering transport mechanisms and increasing
RBC destruction.
| |
ACKNOWLEDGMENT |
We acknowledge the help of Nazim Fataliev's competent purification of
hemoglobin S-Oman, and to all those in the Sultan Qaboos Royal Hospital
who helped in securing many valuable samples.
| |
FOOTNOTES |
Submitted March 16, 1998;
accepted July 16, 1998.
Supported by National Institutes of Health Grants No. HL38655 and
HL55435, and by grants from the European Union (TS3-CT93-0244 DG12HSMU)
to R.K.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ronald L. Nagel, MD, Irving D. Karpas
Professor of Medicine, Albert Einstein of Medicine, 1300 Morris Park
Ave, U921, Bronx, NY 10461; e-mail: nagel{at}aecom.yu.edu.
| |
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Abstract
Introduction