Download Hb Malmö [ß-97(FG-4)His]Gln] leading to polycythemia in a

Ann Hematol (1996) 73 : 183–188
Q Springer-Verlag 1996
ORIGINAL ARTICLE
P. C. Giordano 7 C. L. Harteveld 7 A. Brand
L. N. A. Willems 7 H. C. Kluin-Nelemans 7 R. J. Plug
D. N. Batelaan 7 L. F. Bernini
Hb Malmö [b-97(FG-4)His]Gln] leading to polycythemia
in a Dutch family
Received: 3 June 1996 / Accepted: 5 June 1996
Abstract We have examined six individuals from a
two-generation Dutch family for a suspected hemoglobin (Hb) abnormality. The propositus presented with
polycythemia and complained of persistent weakness,
headache, and epistaxis. All family members initially
showed a normal Hb-electrophoretic pattern, but on
isoelectric focusing, three of them displayed a fast-moving band associated with high packed red cell volumes
(PCV) and increased red blood cell count. The Hb mutant was analyzed at the DNA level by specific gene
fragment amplification (PCR), followed by direct DNA
sequencing, and the mutation was confirmed by restriction enzyme analysis. We found a C]G transversion
(CAC]CAG) at codon 97 of the b-chain, which corresponded to the His]Gln amino acid substitution previously described as Hb Malmö. We report here the
clinical history of the patient, the effects of phlebotomy
treatment, and the effect of subnormal iron conditions
on the erythropoietic recovery after phlebotomy. The
mechanism responsible for the induction of the higher
oxygen affinity is discussed, as are some aspects concerning the occurrence, pathology, treatment, and the
genetic risk of Hb variants with high O2 affinity.
Key words Hb Malmö 7 Hemoglobinopathies 7
Abnormal hemoglobin 7 Polycythemia 7 Oxygen
affinity
P. C. Giordano (Y) 7 C. L. Harteveld 7 R. J. Plug
N. Batelaan 7 L. F. Bernini
Institute of Human Genetics, State University of Leiden,
Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
A. Brand 7 L. N. A. Willems
Department of Hematology, University Hospital Leiden,
Rijnsburgerweg 10, 2333 AL Leiden, The Netherlands
H. C. Kluin-Nelemans
Department of Pulmonology, University Hospital Leiden,
Rijnsburgerweg 10, 2333 AL Leiden, The Netherlands
Introduction
Moderately or nonsymptomatic abnormal Hb traits
which are electrophoretically silent are often recognizable only when more sophisticated methods such as
isoelectrofocusing (IEF), Hb chain fast protein liquid
chromatography (FPLC), high-performance liquid
chromatography (HPLC), or DNA sequencing are applied. An example is the HPLC monitoring of the glycated hemoglobin fraction (Alc) in blood samples of
diabetes mellitus patients, by which unsuspected abnormal hemoglobins are often detected [10]. We describe
here the identification of an electrophoretically silent
mutant by IEF and DNA analysis in a 31-year-old
Dutch man suffering from a significant chronic polycythemia.
Materials and methods
Lithium heparin and Na2-EDTA blood samples were collected.
The DNA extraction was done by selective lysis [29] and high salt
extraction [16]. Red cell indices were obtained from a semiautomatic counter (Sysmex F300, Toa Medical Electronics, Cobe).
Oxygen saturation values were measured in arterial or venous
blood collected in sodium heparin. Aliquots of 3 ml of blood were
directly analyzed using the model 1312 Automatic Blood Gas
Manager (Instrumentation Laboratory, Usselstein). Red cell lysates were examined on starch gel electrophoresis at pH 8.6 [23]
and on isoelectrofocusing, as previously described [9]. The Hb A2
fraction was estimated by ion-exchange column chromatography
[1]. The amount of abnormal Hb component was established by
measuring the optical absorbance of the abnormal versus normal
fractions extracted from the IEF acrylamide gel. The isopropanol
test was used for determination of the Hb stability on the total
hemolysate [5]. The Hb-F percentage was determined by alkaline
denaturation [2]. The in vitro synthetic ratio between the a- and
non-a-globin chains was measured using a method based on
standard procedures [27]. The DNA analysis of the b-globin gene
was done by polymerase chain reaction (PCR), followed by direct
sequencing [11]. The PCRs were carried out in a forced water circulation thermocycler [8]. Sequencing was performed according
to Sanger et al. [22] on PCR products by direct solid-phase sequencing using magnetic beads as a solid support [12]. The FITClabeled M13-sequencing primer was included in the Autoread kit
184
(Pharmacia, Uppsala). Sequence electrophoresis was carried out
according to the standard procedure for the Automated Laser
Fluorescent DNA Sequencing apparatus (A.L.F., Pharmacia,
Uppsala). The gels (6% acrylamide in 7 M urea) were run for 8 h
at 1500 V, 44 mA, (40 W) at 45 7C and scanned by a 5 mW laser
beam. The molecular defect was confirmed by restriction fragment analysis of amplified DNA using BbrPI and PstI endonucleases, as specified in Fig. 4.
Results
Case history. The propositus, a 31-year-old Caucasian
man from a Dutch family, presented at the age of 25
with polycythemia (Hb 19.7 g/dl, RBC 6.33!10 12/l,
PCV 0.56 l/l) and normal white cell and platelet counts.
The bone marrow showed no signs of myeloproliferative hematopoiesis but an extremely active erythropoiesis. The blood volume was increased to 6296 ml (expected value: 5150 ml) with a normal plasma volume of
39 ml/kg, as a consequence of the increased red cell volume of 43 ml/kg (expected value: 30 ml/kg). Secondary
causes of erythrocytosis (sarcoidosis, pheochromocytoma, hepatoma, polycystic kidney disease, von HippelLindau syndrome or CO intoxication) were excluded.
The spirometric parameters, as well as the chest X-ray,
were normal. PCV values were reduced to 0.52 l/l by
phlebotomy, but the patient unfortunately neglected to
have regular follow-up examinations. Five years later,
he had to be urgently admitted to the hospital for a severe persistent epistaxis with an Hb of 21.1 g/dl and a
PCV of 63 l/l. Phlebotomy was again undertaken. Arterial blood gas analysis gave a normal total O2 saturation (sO2) of 97%, but the venous sO2 (at pH 7.4 and
Fig. 1 Oxyhemoglobin dissociation curve from a normal adult individual (——) and from an Hb Malmö heterozygous patient (....)
at pH 7.4 and 37 7C
Fig. 2 IEF diagram showing the mobility of Hb Malmö on a 0.1mm scale in comparison with the mobility of the normal Hb fractions and Hb J-Baltimore
37 7C) was 64.9% at 21 mmHg and the P50 (50% saturation) was obtained at only 14 mmHg (normalp26.5
mmHg). The patient and his family were then investigated further.
Hematological analysis. The hematological parameters
and the sO2 curve (Fig. 1) were strongly suggestive of
the presence of a hemoglobin mutant with increased
oxygen affinity, but with starch gel electrophoresis at
pH 8.6 no abnormal bands were detected. With isoelectrofocusing, however, a fast-moving band representing
47% of the total Hb content was visible at the position
indicated in Fig. 2.
A prolonged starch gel electrophoresis, run at the
standard pH, revealed a slightly faster fraction, emerging just above the Hb A band. The hematological data
of six individuals of the two-generation family are summarized in Table 1.
Molecular analysis. The assortment of the sequence
primers (Fig. 3) was selected in order to cover the complete b-globin gene, including promoter region and
downstream regulatory elements (P200/c1700). Sequencing gaps were avoided by spacing the oligonucleotides in such a way that the sequence obtained with
each primer overlapped with the sequence of the preceding one. Direct sequencing of the PCR fragment obtained by M13-III c BioM primers revealed heterozygosity for a CAC-CAG mutation at codon 97 (Table 2),
which changes the histidine residue into a glutamine.
The C]G transversion at codon 97 disrupts a BbrPI
restriction site present in the wild-type allele and generates a novel PstI restriction site in the mutated allele.
Fig. 3 Schematic representation of the b-globin gene with the location of the oligonucleotides M13-III and BioM designed for
amplification and direct sequencing of the gene fragment
185
The confirmation of the mutant by restriction enzyme
analysis on the DNA fragment obtained by amplification with M13-III and aPCO4 primers is shown in Fig.
4.
Discussion
Structure and oxygenation mechanism
Fig. 4 Schematic representation of the b-globin gene with the location of the amplification primers M13-III (see Fig. 3) and
caPCO4 (5b-GGTGAACGTGGATGAAGTT-3b) and the location of the codon 97 point mutation corresponding with the restriction site of BprPI and PstI endonucleases. The DNA fragment obtained by amplification and digestion is shown on the two
gels. Due to the disruption of the BbrPI restriction site caused by
the mutation, the two normal smaller segments generated by this
enzyme appear on the left-hand gel, in association with the nondigested amplification fragment coming from the mutated allele
(left to right; lane 1 the undigested PCR fragment; lanes 2, 4, 6 the
two normal fragments generated by BprPI; lanes 3, 5, 6 the same
fragments together with the undigested one in the carriers of Hb
Malmö). After digestion with PstI which cuts at the new restriction site generated by the mutation, the same heterozygous pattern is observed on the right-hand gel in the mutation carriers
(lanes 2, 4, 6), while the homozygous wild types (lanes 1, 3, 5)
remain undigested
Many hemoglobin mutants with increased oxygen affinity have been described at different positions of the globin chains, and various mechanisms have been proposed to explain the modification of the normal physiological function of the molecule. Some amino acid
substitutions are known to influence the heme pocket
environment, e.g., Hb Zürich (b-63 His]Arg). Mutations at the carboxy terminal of the b chains (b-146) are
known to alter the Bohr effect; mutations at both the
amino and carboxy ends alter salt bridges and 2,3-DPG
binding.
The great majority of the abnormal hemoglobins described between residues 89 and 109 of the b-globin
chain are associated with an increased oxygen affinity,
mostly as a result of modifications in the a1/b2-subunit
contact which shift the equilibrium of the quaternary
structure in favor of the oxygenated (R) state. Histidine
at position b-97 is an evolutionary invariant residue located at the interface of the b2/a1 chains and therefore
involved in the changes which take place during the
oxygenation/deoxygenation of the tetramer. This residue, in the deoxy form, is situated in front of the a-C
helix between the a-44 (proline) and the a-41 (threonine) residues. During the oxygenation process, the
dimers a1/b1 and a2/b2 converge 157 toward each other
and the b-97 residue moves along the aC helix, ending
between the two threonine residues a-41 and a-38.
Table 1 Hematological data of the family members (N normal, * propositus after phlebotomy, P negative, c positive, Hp haptoglobin, – – not measured)
Individuals
Sex/age
I-1
M56
I-2
F58
II-1
M36
II-2
M35
II-3
M31*
II-4
M27
Hb (g/dl)
PCV (l/l)
RBC (10 12/l)
MCV (fl)
MCH (pg)
MCHC (g/dl)
Hb A2 (%)
Hb F (%)
Hb X (%)
b/a ratio
Hp (mg/dl)
Ferritin (mg/l)
Inclusions
Hb instability
Osm. frag.
15.3
0.45
4.60
97.0
33.2
34.0
2.42
1.4
–
––
55
––
––
–
N
18.0
0.54
6.13
87.0
29.4
33.3
2.55
~1
48
––
34
138
–
–
N
14.3
0.43
4.71
91.0
30.4
33.2
2.98
~1
–
––
55
––
––
–
N
15.3
0.45
4.60
97.0
33.2
34.0
2.42
~1
–
––
61
––
––
–
N
13.7
0.47
7.30
64.0
19.2
29.1
2.21
~1
47
1.25 a
56
5
–
–
N
18.7
0.55
6.17
88.0
30.3
34.0
3.0
~1
48
––
24
––
–
–
N
a
The moderate imbalance in the (b/a) synthetic ratio measured in the propositus after in vitro incubation in the presence of labeled
leucine is a known artifact, often observed in the presence of iron deficiency. No a-thalassemia defects were found
186
Table 2 Sequence of the bFG p-helix in HbA and Hb
Malmö. The heme-linked and
H-bonded residues are indicated, as well as the restriction
sites recognized by the BbrPI
and PstI endonucleases
Codon number
Helix position
92 93
F8 F9
b Normal
GAC AAG
h
His Cys Asp Lys
GAC AAG
h
His Cys Asp Lys
–––
–––
b Malmö
BbrPI
PstI
94
FGl
95
FG2
96
97
FG3 FG4
CTG
Leu
CTG
Leu
–––
CTG
98
FG5
99
Gl
CAC GTG :
His@ Val : Asp
CAG GTG :
Val : Asp
Gln
CAC*C GTG– – – –
–––
––
A*G
DNA sequence
Amino acid sequence
DNA sequence
Amino acid sequence
Restriction site
Restriction site
h p Heme link (His b2 is a heme-linked residue).
: p H bond (CuO of main chain of val b2-98 is H-bonded with Tyr b2-145, which is a a1/b2 interface contact with Thr a1-41 in deoxyhemoglobin; Asp b2-99 is H-bonded with Tyr a1-42 and
Asn a1-97 in deoxyhemoglobin).
@ p Intra helix contact (His FG4 is hydrogen-bonded through a water molecule to the C terminus of
the FG1-FG4 helix).
* p Endonuclease cutting sites used in DNA restriction-site analysis.
Since histidine b-97 is directly involved neither in
the hydrogen bonds nor in salt bridges or packing contacts between the b2/a1-subunits, several models have
been proposed to explain the increased stability of Hb
Malmö in the oxygenated (R) state.
Perutz et al. [21] studied the involvement of the b-97
histidine residue in the Bohr-effect mechanism and
concluded that a contribution of this residue to the
Bohr effect could be claimed in des-His HbA-CO
(b146 histidine-cleaved HbA-CO) but not in normal
HbA. They also measured pKa values for histidine at
position b-97 which were found to be significantly higher (7.8) than that expected in nonbonded histidine, and
they attributed this higher value to the interaction between the imidazole of histidine FG4 and the C-terminus of the FG1-FG4 helix, to which histidine is hydrogen-bonded through a water molecule.
In Hb Malmö and also in the other three hemoglobin variants described at position b-97, i.e., Hb Wood
(His]Leu) [25], Hb Nagoya (His]Pro) [18], and Hb
Moriguchi (His]Tyr) [19], the substitutions disrupt the
intra-helix contact of the conserved histidine, which is
probably essential for maintenance of the right shape of
the b-FG p-helix. The stability of this helix, which is
the closing gate of the heme pocket, is maintained in
the deoxy state by several molecular contacts (Table 2):
the H-bonds between Asp b2-99, Tyr a1-42 and Asn a197; the heme-linking F9 histidine residue at position b92; the H-bond between the CuO group of val b2-98
main chain and the Tyr b2-145 residue, whose side
chain is also a a1/b2 interface contact with Thr a1-41.
This shows that a structural modification at a single
site, depending upon the location, the bulk, and the
chemical nature of the amino acid substitution, can
have far-reaching consequences for the stability and
function of hemoglobin.
Occurrence
To the best of our knowledge, the family described in
our study is the fifth report of polycythemia induced by
Hb-Malmö and the first in the Dutch population. The
family claims Dutch descent and has no knowledge of
Scandinavian ancestors.
The Hb Malmö cases described previously were all
in association with polycythemia. The first case was
seen by Lorkin and Lehman in 1970 in a four-generation Caucasian Swedish family [15]. The second report
was of a large American family of English ancestry [3].
In this report, the physiological properties of the mutant were extensively described showing an increased
sO2, a normal Bohr effect, and a decreased heme-heme
interaction. Subsequently, the mutant was found in a
French patient who was not ethnically specified. Another case [10] was described in Sicily, in a man of
northern European appearance. It is interesting to note
that this island was under Norman domination for a
long period of time.
In order to change the histidine codon (CAC) at position b-97 into a glutamine codon, a transversion of the
third pyrimidine to either adenine or a guanine is
needed. Recently, a second Swedish Hb Malmö family
has been described in which the amino acid substitution
His]Gln at position b-97 was coded by a CAA triplet
[14]. Investigation at the DNA level in one of the carriers from the first Swedish family revealed that the
same His]Glu substitution at position b-97 was coded
by a CAG codon. This result implies that two different
rare mutation events occurred independently in the
same population, causing the same amino acid substitution.
Hematology, pathology, treatment and genetic risk
In carriers of Hb variants with high oxygen affinity, the
primary physiological response to the deficient oxygen
delivery to the tissues is represented by an erythropoietin-mediated erythrocytosis, while increased cardiac
output and local modifications of the blood flow in peripheral tissues may contribute to a better oxygen unloading in vital organs. The great majority of affected
individuals can tolerate this condition very well, but a
few can experience problems due to hyperviscosity of
the blood. In the latter case, the therapeutic dilemma is
187
whether and to what extent it is convenient to decrease
the red cell mass by phlebotomy [24].
The role of phlebotomy as a therapeutic tool to reduce viscosity in polycythemia induced by high O2 affinity Hb mutants is debatable [24]. It has been postulated by Wade et al. [26] that increased blood flow reduces the risk of thrombosis in patients with Hb Yakima (a high O2 affinity mutant induced by a substitution
Asp]His at position b-99). Individual cases of vascular
obstructions or transitory brain ischemia in the presence of high oxygen affinity Hb mutants have been reported [7, 25, 28, 30], but substantial data on this matter
are limited.
In the present case, the mother of the propositus (I2) experienced a transient ischemic attack at the age of
27, after which she was regularly phlebotomized until 5
years ago. After phlebotomy treatment, the propositus
himself experienced an improvement of his general
condition and the disappearance of complaints. Comparing the hematological data of the three carriers (I-2,
II-3, II-4), we observed that after phlebotomy treatment the propositus (II-3) had not only a low-normal
Hb concentration and normal PCV, but also low MCV
and MCH values as a consequence of iron deficiency.
It would seem logical to assume that after phlebotomy, the lowered Hb values and the altered physiology
of the mutant would account for a reduced oxygen release to the tissues. The compensatory erythropoietic
activity in the presence of hypoxia increases the output
of young 2,3-DPG-rich red cells, however, with an enhanced oxygen release behavior. A similar positive experience was reported in other carriers of high O2 affinity mutants [6] after reduction of PCV and induction of
a moderate iron deficiency by phlebotomy. Nute et al.
[17] reported a family with a b-globin mutant with increased O2 affinity (Hb Olympia, b-20 Val]Met),
where one carrier had a spontaneous reduction of erythrocytosis with a co-existing iron deficiency. Conversely, in a patient with another high O2 affinity mutant
(Hb Osler b-145 Tyr]Asp), decreased Hb levels
brought about by phlebotomy treatment led to a diminished anaerobic threshold [4].
Repeated phlebotomy induces iron deficiency and
decreases of MCH and MCV. At any given PCV value,
whole-blood viscosity is not influenced by the volume
and hemoglobin content of red cells [20]. However, at
identical packed red cell volumes, patients with a low
MCH have also lower Hb and oxygen-carrying capacity. This is likely to be detrimental in a hypoxic state and
possibly critical under sudden physical stress. The effect
of smoking should also be taken into consideration,
since as much as 10% of the available hemoglobin
could be irreversibly transformed into nonfunctional
CO-Hb by cigarette smoke.
In conclusion, if PCV reduction becomes necessary
because of a threatening blood hyperviscosity it should
be undertaken with caution, and the tolerance of patients, particularly the elderly, should be monitored by
exercise testing. Nonetheless, a decrease of PCV to-
gether with a moderate and monitored iron depletion
seems to be justified to avoid severe circulatory accidents in patients with symptomatic polycythemia due to
high O2 affinity hemoglobinopathies.
The chance of finding uncommon Hb mutants in homozygous conditions, except in some rare consanguineous cases, is extremely low. Sometimes, however, it is
possible to find uncommon abnormal b-globin mutants
in compound heterozygous conditions combined with
b-thalassemia, which is a much more frequent hemoglobin defect, which eliminates the expression of the affected b-gene and thus creates a homozygous-like phenotype for the rare allele present on the homologous
chromosome.
Although one could predict severe pathology in a
compound heterozygous condition such as b-thalassemia/Hb Malmö, the only case described with this combination, the Sicilian patient reported by Girino et al.
[10], showed only minor circulatory problems (i.e., varicose veins at the age of 13). Despite the extreme hematological parameters (Hb 23 g/dl, RBC 10.5!10 12/l,
PCV 0.90 l/l) and a 4.5-fold increased erythropoiesis,
this patient had reached, at the time of the report, the
age of 27, was being intensively treated with phlebotomy (to maintain the PCV at 60–65%), and was living a
normal working life without experiencing ischemic episodes.
In the search for compounds that might prevent the
aggregation of hemoglobin S in patients with sickle cell
anemia, two anti-lipidemic drugs were found which induced the opposite effect, being very strong stabilizers
of the deoxyhemoglobin conformation [13]. The drugs
(bezafibrate derivates) reduced the oxygen affinity of
the hemoglobin molecule very effectively, without damaging the erythrocytes. Unfortunately, however, these
drugs are tightly bound by serum albumin at the physiological concentrations present in the serum. This prevents their effect on the conformation of the Hb tetramer and an eventual pharmaceutical application, at
least for the time being, in the treatment of polycythemia induced by high oxygen Hb variants.
References
1. Bernini LF (1969) Rapid estimation of hemoglobin A2 by
DEAE chromatography. Biochem Genet 2 : 305–310
2. Betke K, Marti HR, Schlicht I (1959) Estimation of small percentages of foetal haemoglobin. Nature 184 : 1877–1878
3. Boyer SH, Charache S, Fairbanks VF, Maldonado JE, Noyes
A, Gayle EE (1972) Hemoglobin Malmo b97 (FG4)
His]Gln: a cause of polycythemia. J Clin Invest 51 : 666–676
4. Butler WM, Spratling L, Kark JA, Schoomaker EB (1982)
Hemoglobin Osler: report of a new family with exercise studies before and after phlebotomy. Am J Hematol 13 : 293–301
5. Carrell RW, Kay R (1972) A simple method for the detection
of unstable haemoglobins. Br J Haematol 23 : 615–619
6. Charache F, Achuff S, Winslow R, Adamson J, Chervenick P
(1978) Variability of the homeostatic response to altered P50.
Blood 52 : 1156–1162
7. Fairbanks VF, Maldonado JE, Charache S, Boyer SH (1971)
Familial erythrocytosis due to electrophoretically undetecta-
188
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
ble hemoglobin with impaired oxygen dissociation (hemoglobin Malmo, a2b2 97 gln). Mayo Clin Proc 46 : 721–727
Giordano PC, Fodde R, Losekoot M, Bernini LF (1989) Design of a programmable automatic apparatus for the DNA
polymerase chain reaction. Technique 1 : 16–20
Giordano PC, Harteveld CL, Kok PJMJ, Geenen A, Batelaan
D, Amons R, Bernini LF (1996) Hb Gouda [a72(EF1)
His]Gln] a new silent a-chain variant. Hemoglobin 20 : 21–
29
Girino M, Riccardi A, Mosca A, Paleari R, Bonomo P (1989)
Double heterozygosity for Hb-Malmo fb97(FG4)His]Gln]
and b-thalassemia traits. Haematologica (Pavia) 74 : 187–90
Harteveld CL, Losekoot M, Haak H, Heister JGAM, Giordano PC, Bernini LF (1994) A novel polyadenylation signal mutation in the a2-globin gene causing a-thalassemia. Br J Haematol 87 : 139–143
Hultman T, Ståhl S, Hornes E, Uhlin M (1989) Direct solidphase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res 17 : 4937–
4946
Lalezari I, Lalezari P, Poyart C, Marden M, Kister J, Bohn B,
Fermi G, Prutz MF (1990) New effectors of human hemoglobin: structure and function. Biochemistry 29 : 1515–1523
Landin B, Berglund S, Walman K (1996) Two different mutations in codon 97 of the b-globin gene cause Hb Malmö in
Sweden. Am J Hematol 51 : 32–36
Lorkin PA, Lehman H (1970) Two new pathological haemoglobins: Olmsted b141 (H19) Leu leads to Arg and Malmo
b97 (FG4) His leads to Gln. Biochem J 119 : 68P
Miller SA, Dykes DD, Polesky HF (1988) A simple saltingout procedure for extracting DNA from human nucleated
cells. Nucleic Acids Res 16 : 1215
Nute PE, Pierce HI, Hermodson MA, Adamson J, Stammatoyannopoulos G (1978) Environmental modification of gene
expression: iron deficiency masks erythrocytosis in a female
with hemoglobin Olympia. Hemoglobin 2 : 275–279
Ohba Y, Imanaka M, Matsuoka M, Hattori Y, Miyaji T, Funaki C, Shibata K, Shimokata H, Kuzuya F, Miwa S (1985) A
new unstable, high oxygen affinity hemoglobin: Hb Nagoya
or b97(FG4)His]Pro. Hemoglobin 9 : 11–24
19. Ohba Y, Imai K, Kumada I, Ohsawa A, Miyaji T (1989) Hb
Moriguchi or a2b297(FG4) His]Tyr substitution at the a1b2
interface. Hemoglobin 13 : 367–376
20. Pearson TC, Grimes AJ, Slater NGP, Wetherley-Mein G
(1981) Viscosity and iron deficiency in treated polycythaemia.
Br J Haematol 49 : 123–127
21. Perutz FM, Gronenborn AM, Clore GM, Fogg JH, Shih DT
(1985) The pKa values of two histidine residues in human hemoglobin, the Bohr effect, and the dipole moments of alphahelices. J Mol Biol 183 : 491–498
22. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing
with chain terminating inhibitors. Proc Natl Acad Sci USA
74 : 5463
23. Smithies O (1965) Characterization of genetic variants of
blood proteins. Vox Sang 10 : 359–369
24. Stammatoyannopoulos G, Bellingham AJ, Lenfant C, Finch
CH (1971) Abnormal hemoglobins with high and low oxygen
affinity. Annu Rev Med 22 : 221–234
25. Taketa F, Huang YP, Libnoch JA, Dessel BH (1975) Hemoglobin Wood b97 (FG4) His]Leu. A new high-oxygen-affinity hemoglobin associated with familial erythrocytosis. Biochim Biophys Acta 400 : 348–353
26. Wade JPH, du Boulay GH, Marshall J, et al (1980) Cerebral
blood flow , haematocrit and viscosity in subjects with a high
oxygen affinity haemoglobin variant. Acta Scand Neurol
61 : 210–215
27. Weatheral DJ, Clegg JB (1981) Screening procedures for
quantitative abnormalities in hemoglobin synthesis. Methods
Enzymol 76 : 751–763
28. Weatheral DJ, Clegg JB, Callender ST et al (1977) Haemoglobin Radcliffe: a high oxygen affinity variant causing familial polycythaemia. Br J Haematol 35 : 177–191
29. Weening RS, Roos D, Loos JA (1974) Oxygen consumption
of phagocytosing cells in human leucocyte and granulocyte
preparations: a comparative study. Lab Clin Med 83 : 570–
574
30. White JM, Szur L, Gillies IDS, Lorkin PA, Lehmann H
(1973) Familial polycythaemia caused by a new haemoglobin
variant, Hb Heathrow, b103 (G5) Phe]Leu. Br Med J
3 : 665–667