Hemoglobin, 34(4):383–388, (2010)Copyright © Informa UK Ltd.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630269.2010.486331

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LHEM0363-02691532-432XHemoglobin, Vol. 1, No. 1, Apr 2010: pp. 0–0Hemoglobin

SHORT COMMUNICATION

ERYTHROCYTOSIS DUE TO A COMBINATION OF THE HIGH OXYGEN

AFFINITY HEMOGLOBIN VARIANT, Hb OLYMPIA [b20(B2)VAL®MET]

WITH b- AND a-THALASSEMIA MUTATIONS: FIRST CASE

IN THE LITERATURE

Erythrocytosis Due to Hb Olympia, b0- and a0-ThalassemiaV. Kalotychou et al.

Vassiliki Kalotychou,1 Revekka Tzanetea,1 Kostas Konstantopoulos,1

Ioannis Papassotiriou,2 and Ioannis Rombos1

1First Department of Internal Medicine, University of Athens School of Medicine, Laikon Hospital, Athens, Greece2Department of Clinical Biochemistry, Aghia Sophia Children’s Hospital, Athens, Greece

� A 40-year-old Greek male was admitted to the hospital because of acute respiratory infection.The patient has been undergoing regular venesection for erythrocytosis for 20 years; he has also beentaking oral anticoagulants for thrombosis for 15 years. The molecular defect for erythrocytosis wasdetected together with the rare Hb Olympia (HBB:c.61G>A) variant. This hemoglobin (Hb) variantwas found in combination with two thalassemia-type globin gene defects, namely b0-thalassemia(b0-thal), HBB:c.118C>T and a0-thal (– –MED). This combination of three molecular defects is thefirst such case reported in the literature.

Keywords Hb Olympia, Erythrocytosis, β0-Thalassemia (β0-thal), α0-Thalassemia (α0-thal)

A 40-year-old Greek non smoking male presented at the outpatientclinic complaining of dizziness, fainting, tinnitus, headaches and dyspnoea.Following clinical examination and relevant laboratory tests he was admit-ted due to a respiratory infection. On admission, the following findingswere observed: moderate dyspnoea, remarkable kyphoscoliosis, abdominaldistention, telangectasias at thorax and abdomen, no jaundice. No otherclinical findings were noticed.

Received 30 November 2009; Accepted 29 January 2010.Address correspondence to Vassiliki Kalotychou, BSci, PhD, First Department of Internal Medicine,

University of Athens, School of Medicine, Laikon Hospital, 17 Agiou Thoma Str, 11527, Athens, Greece;Tel: +210-745-6240; Fax: +210-778-8830; E-mail: vkalotyc@med.uoa.gr

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384 V. Kalotychou et al.

His past medical history revealed that when he was 17 years old, a highhematocrit [PCV (packed cell volume) (L/L)] level of 0.64 was firstobserved in routine tests. As far as we know, it was suggested that moreinvestigation was needed but no tests were done. For the first few years notreatment was offered but after 3 years, the patient constantly presentedwith a red face and red conjunctives, many times being cyanotic, scratchingafter a hot bath, headaches, weakness and pain in both legs.

Although the signs and symptoms associated with erythrocytosis arenot specific, the above clinical manifestations were attributed to high PCV(0.64 L/L) values and the patient was started on a venesection protocol,removing 360 mL peripheral blood, three times per year. Despite this treat-ment, when he was 25 years old, a thrombosis at the level of the subclavian-brachial vein occurred and he was put on anticoagulants. At the age of 28he was diagnosed as having atrial fibrillation and since then has been onmedication.

On admission to our hospital no abnormalities from other blood cellseries or splenomegaly were detected; no evidence of a myeloproliferativedisorder existed. Cardiac echo-test and heart-radiology were negative forsize or function abnormality. No facilities for measuring erythropoietinlevels were available but an erythropoietin-producing tumor was excludedafter a negative computer tomography (CT) examination of the chest andabdomen.

Hematological data before starting venesection was as follows: redblood cell mass 46.7 mL/Kg BW (body weight), (normal values for men upto 36 mL/Kg BW), RBC 8.8 × 1012/L, PCV 0.62 L/L, Hb 19.8 g/dL, MCV70.5 fL, MCH 22.5 pg, MCHC 31.9 g/dL. Biochemical tests were normal,with normal levels of urea, creatinine and iron. He has followed a regularvenesection protocol for the last 20 years for amelioration of the symptomsof erythrocytosis, as mentioned above.

On reexamination after admission to our hospital, he was iron deficientwith low serum iron and ferritin levels, 26 μg/dL and 4.0 ng/dL, respec-tively, due to phlebotomies targeting to decrease PCV (about 0.50-0.55)thus protecting from thromboses. A recent hematological examinationshowed: RBC 8.75 × 1012/L, PCV 0.54 L/L, Hb 15 g/dL, MCV 62 fL, MCH17.1 pg, MCHC 26.6 g/dL, WBC 5100/mm3, platelets 159.000/mm3.

Peripheral blood morphology revealed red cell crowding, erythrocyto-sis, anisocytosis, microcytosis, poikilocytosis, hypochromia and target cells.Hemoglobin (Hb) electrophoresis on cellulose acetate showed an elevatedA2 fraction (Hb A2 4.5%) and normal Hb F (2%). These findings point to athalassemia state.

Genomic DNA was isolated from peripheral blood white cells accordingto a standard protocol (QIAamp DNA Blood Midi kit; Qiagen GmbH,Hilden, Germany) and molecular studies were carried out in order to

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Erythrocytosis Due to Hb Olympia, b0- and a0-Thalassemia 385

screen the β globin gene. Amplification refractory mutation system (ARMS)was performed and the HBB:c.118 C>T mutation was detected. The pres-ence of this thalassemia-specific mutation was verified by sequencing of theβ-globin gene. A fragment extending from −239 to +632 bp relative to theCap site was amplified by using the following pairs of primers: (forward)5’-TTT AGT AGC AAT TTG TAC TG-3’, reverse: 5’-CAC TGA TGC AATCAT TCG TC-3’.

The reaction mixture (100 μL) for the amplification of the β-globingene fragment contained 30 pmoles of each primer, 2.5 mM MgCl2, 200 μMof each dNTP, 10 mM Tris-HCl pH 8.4, 50 mM KCl and 2 U of Taq DNApolymerase (Invitrogen, Carlsbad, CA, USA). The polymerase chain reac-tion (PCR)-conditions were as follows: denaturation for 1 min. at 94°C,annealing for 1 min. at 55°C and primer extension for 1 min. at 72°C, 30cycles.

Moreover, sequencing analysis confirmed the presence of the HBB:c.118C>T mutation (Figures 1a and 1b) and detected an additional point muta-tion in the coding region of the β-globin gene, namely HBB:c.61 G>A lead-ing to a substitution of valine with methionine (Figures 2a and 2b). TheG>A mutation is the molecular defect corresponding to Hb Olympia, a highoxygen affinity Hb [β20(B2)Val→Met], explaining the increased levels ofRBC mass, Hb and PCV of the proband.

Furthermore, his sister was examined and the following hematologicalindices were revealed: RBC 5.77 × 1012/L, PCV 0.35 L/L, Hb 11.5 g/dL,MCV 60.8 fL, MCH 19.9 pg, MCHC 32.8 g/dL. Hemoglobin electrophoresisshowed an elevated A2 fraction (Hb A2 4.4%). Genomic DNA was isolatedfrom her peripheral blood white cells and molecular study (ARMS) was per-formed using the appropriate primers for the HBB:c.118C>T mutation anddirect sequencing as described above for the proband. Sequencing analysisexcluded the presence of Hb Olympia. According to these family data theHBB:c.61G>A mutation resides in trans with the HBB:c.118C>T mutation.

We estimated the P50 value (the partial oxygen pressure at which Hb ishalf-saturated with oxygen). This value was found to be 17.5 mmHg (theoxygen equilibrium curve is shifted to the left), whereas in normal red cellsand under normal conditions, P50 is 26 mmHg. Intra-cellular 2,3-DPG(2,3-diphosphoglycerate), was 4.32 μM/mL RBC (normal values 4.79 ± 0.20μΜ/mL RBC).

The α- to β-globin chains ratio was estimated by biosynthesis for afurther confirmation of the thalassemia-related chain imbalance. Surpris-ingly, this α to β ratio was found to be balanced, despite the (expected)lower levels of β-globin gene chains. This finding led us to the suspicion ofthe coexistence of an α-globin gene defect. Further molecular studies usingPCR-based methods identified the presence of an α-globin gene mutation(– –MED) (1).

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386 V. Kalotychou et al.

Hb Olympia may be masked on electrophoresis or cellulose acetate atalkaline pH as it migrates together with Hb A, being an electrophoreticallyand chromatographically “silent” variant (2). In Hb Olympia, it is possiblethat the hydrophobic side chain of methionine (instead of valine) mayprotrude into the interior of the subunit or that the bulk of this side chain,containing a relatively large sulfur atom, may distort the overall configura-tion of this portion of helix B, thus affecting other sites having a role in sub-unit interactions (2).

Several cases of this high oxygen affinity Hb, with or without β-thalassemia(β-thal) have been described. In France, Hb Olympia was found in at leastnine unrelated families (3,4), three of them being of Mediterranean origin. Inthese cases, seven individuals were heterozygous for Hb Olympia and they had

FIGURE 1 (a) Nucleotide analysis of exon 2 of the β-globin gene with a forward primer (5’-TTT TAGTAG CAA TTT GTA CTG-3’). An arrow shows the HBB:c.118 C>T mutation. (b) Nucleotide analysis ofexon 2 of the β-globin gene with a reverse primer (5’-CAC TGA TGC AAT CAT TCG TC-3’). An arrowshows the HBB:c.118 C>T mutation.

A

B

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Erythrocytosis Due to Hb Olympia, b0- and a0-Thalassemia 387

a well-tolerated erythrocytosis (RBC 5.2–6.5 × 1012/L, PCV 0.48–0.54 L/L,MCV 88–91 fL), while three compound heterozygotes for the rare Hb vari-ant and a β-thalassemic defect suffered from a more severe disease (RBC7.7–9.0 × 1012/L, PCV 0.58–0.59 L/L, MCV 60–72 fL).

The conventional treatment to polycytosis is myelosuppresive medica-tion but this may lead to secondary malignancies. On the other hand, inolder patients erythrocytosis is usually complicated by thromboses. Havingthis in mind, the detection of high oxygen affinity Hbs is substantial (5,6).In the proband erythrocytosis was ameliorated with regular venesectionstwice per year, and the ensuing complications were treated with anticoagu-lants and antiarrythmics.

FIGURE 2 (a) Nucleotide analysis of exon 1 of the β-globin gene with a forward primer (5’-TTT TAGTAG CAA TTT GTA CTG-3’). An arrow shows the HBB:c.61G>A mutation. (b) Nucleotide analysis ofexon 1 of the β-globin gene with a reverse primer (5’-CAC TGA TGC AAT CAT TCG TC-3’). An arrowshows the HBB:c.61G>A mutation.

A

B

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388 V. Kalotychou et al.

Our case seems to be an uncommon combination of three differentmolecular defects within the globin gene clusters. Two reside in trans to theβ-globin gene cluster and the third to the α-globin gene cluster. Further-more, it is important to precisely investigate (with DNA studies) the β0-thalcarriers when counseling for prenatal diagnosis, thus for excluding theα0-thal co-inheritance.

Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of this article.

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2. Stamatoyannopoulos G, Nute PE, Adamson JW, Bellingham AJ, Funk D. Hemoglobin Olympia(20 valine leads to methionine): an electrophoretically silent variant associated with high oxygenaffinity and erythrocytosis. J Clin Invest. 1973;52(2):342–349.

3. Wajcman H, Galacteros F. Abnormal hemoglobins with high oxygen affinity and erythrocytosis.Hematol Cell Ther. 1996;38(4):305–312.

4. Wajcman H, Galacteros F. Hemoglobins with high oxygen affinity leading to erythrocytosis. Newvariants and new concepts. Hemoglobin 2005;29(2):91–106.

5. Dash S, Das R. Late emergence of polycythemia in a case of Hb Chandigargh [β94(FG4)Asp→Gly].Hemoglobin 2004;28(3):273–274.

6. Kister J, Baudin-Creuza V, Kiger L, et al. Hb Montfermeil [β130(H8)Tyr→Cys]: suggests a key rolefor the interaction between helix A and H in oxygen affinity of the hemoglobin molecule. BloodCells Mol Dis. 2005;34(2):166–173.

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