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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. 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