Hemoglobin, 33(6):498–502, (2009)Copyright © Informa UK Ltd.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630260903341313

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LHEM0363-02691532-432XHemoglobin, Vol. 33, No. 6, Sep 2009: pp. 0–0Hemoglobin

SHORT COMMUNICATION

HB ALPERTON [b135(H13)ALA®VAL] SHOWS DECREASED

OXYGEN AFFINITY

Hb Alperton [b135(H13)Ala→Val]K. Zurbriggen et al.

Karin Zurbriggen,1 Marlis Schmid,1 Markus Schmugge,1

Heinz Troxler,2 and Oliver Speer1,3

1Division of Haematology, University Children’s Hospital, Zürich, Switzerland2Division of Clinical Chemistry and Biochemistry, University Children’s Hospital, Zürich, Switzerland3Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland

� A 14-year-old male child presented with microcytosis, a known a+-thalassemia (a-thal) heterozy-gote and a hemizygous glucose-6-phosphate dehydrogenase (G6PD) deficiency. Furthermore, cationexchange high performance liquid chromatography (HPLC) revealed an additional peak elutingslightly before Hb A. The peak area of the variant was equal to that of Hb A, suggesting a b-globinvariant. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis confirmed the mutation at the protein level. The variant was detectable by isoelectricfocusing (IEF) or by reversed phase HPLC. DNA sequencing revealed a heterozygous mutation atcodon 135 of the b gene, already described as Hb Alperton. Hb Alperton showed decreased oxygenaffinity. Neither biochemical nor clinical characteristics for Hb Alperton have been reported so far.

Keywords b-Globin variant, PolyCAT A high performance liquid chromatography(HPLC), Mass spectrometry (MS), oxygen equilibrium

A 14-year-old Indian male patient was referred to our out patient clinicfor an unclear microcytosis with sporadic mild anemia. Routine laboratorytests revealed a total hemoglobin (Hb) level in the lower normal range (Hb12.5-14.5 g/dL), microcytosis (MCV 63.0 fL), and borderline values for HbCO (0.6%, normal <1.0%) and bilirubin (20 μmol/L, normal <17 μmol/l).Ferritin levels were normal (Table 1).

Received 14 April 2009; Accepted 5 June 2009.Address correspondence to Dr. Oliver Speer, Division of Haematology, University Children’s

Hospital, Steinwiessstrasse 75, CH-8032 Zurich, Switzerland; Tel: +41-44-266-80-87; Fax: +41-44-266-71-71;E-mail: oliver.speer@kispi.uzh.ch

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Hb Alperton [b135(H13)Ala®Val] 499

With these findings we decided to screen for a possible hemoglobinopa-thy and applied different techniques and methods. Routine cationexchange high performance liquid chromatography (HPLC) on a PolyCATA column was performed as described by Bisse and Wieland (1). For furthercharacterization of the lysate, we applied matrix assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF-MS), reversedphase HPLC on a Vydac 124TP54 C4 column (Bucher Biotech, Basel,Switzerland) and isoelectric focusing (IEF) as described by Kleinert et al.(2), Kutlar et al. (3) and Basset et al. (4), respectively. DNA was preparedfrom peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen,Hombrechtikon, Switzerland). The b-globin gene was amplified using theforward primer (5′-CAT CAG TGT GGA AGT CTC AGG-3′) and the reverseprimer (5′-TTA AAT GCA CTG ACC TCC CAC-3′). Hemoglobin tonometrywas performed with 1.5 mL heparinized whole blood as described by Engeland Duc (5). Blood gas analysis was performed on a Radiometer NPT7blood gas analyzer (Radiometer, Thalwil, Switzerland).

In cation exchange chromatography, the Hb variant was observed as anintense signal eluting slightly before Hb A (Figure 1A). The MALDI-TOFmass spectrum revealed an additional peak at m/z 15896 Da that corre-sponded to the mutant b chain and exhibited a mass shift of +28 Da com-pared to the normal b chain (Figure 1B). The Hb variant was detectedneither by reversed phase HPLC nor by IEF. By sequencing the b-globingene a heterozygous C>T transition at the second nucleotide of codon 135was identified (Figure 2). This GCT>GTT transition results in an Ala→Val

TABLE 1 Hematological Parameters of the Heterozygous Hb Alperton Carrier

Parameters First admissionOxygen affinity

studyNormal values

(range)

Year of analysis 2008 2009 –Sex-Age M-13 M-14 –Hb (g/dL) 12.5 14.3 13.0–17.0RBC (1012/L) 6.08 6.77 4.50–5.50PCV (L/L) 0.39 0.45 0.37–0.50MCV (fL) 63.0 65.9 78.0–100.0MCH (pg) 21.0 21.1 25.0–35.0MCHC (g/dL) 32.5 32.1 31.0–36.0Ferritin (μg/L) 86.6 43.4 10–180Hb F (%) 0.3 0.3 <1.5Hb A2 (%) 2.7 2.7 2.0–3.5Hb A (%) 42.8 40.6 >95.0Hb Alperton (%) 54.2 56.4 –P50 (kPa) n.d. 4.75 3.9–4.7Hill coefficient n.d. 2.84 2.3–2.7Hb CO (%) 0.6 0.1 <1.0

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500 K. Zurbriggen et al.

exchange at position 135 in the b chain, and the calculated mass shift of thevariant chain (+28 Da) corresponded well with the observed shift in MS.The variant was named Hb Alperton.

This Hb variant has already been named (6), however, no clinical orbiochemical data has been presented so far. We therefore decided to ana-lyze the oxygen affinity of Hb Alperton. Hemoglobin tonometry revealed amildly decreased oxygen affinity of the patient’s Hb. The P50 value for the

FIGURE 1 Biochemical analyses of a lysate sample of the Hb Alperton carrier. A) Cation exchangeHPLC reveals an intense signal eluting slightly before Hb A that corresponds to Hb Alperton (Hb X).The glycated species of Hb Alperton (Hb X1C) eluted slightly before the glycated Hb A (Hb A1C). Asexpected, no change in the Hb A2 (a2d2) peak was detected. B) The MALDI-TOF mass spectrum of thelysate. The mutated b chain is assigned bX. Peaks marked with an asterisk (*) correspond to matrixadducts.

FIGURE 2 DNA sequence of the Hb Alperton carrier with the GCT>GTT transition at codon 135 in theb gene. The panel shows the forward sequence. The mutation is marked with an arrow.H

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Hb Alperton [b135(H13)Ala®Val] 501

patient’s whole blood was 4.75 kPa [normal 4.3 + – 0.16 kPa; 95% CI (95%confidence interval) 4.2 – 4.4; n = 19] (Table 1). Hemoglobin stability testswere not performed as the HPLC and MALDI-TOF analysis did indicatethat Hb Alperton is very probably not an unstable Hb variant.

We were unable to perform family studies as the patient is an adoptedchild and the parents are unknown. Furthermore, a heterozygous 3.7 kbdeletion (–a3.7) on the a-globin gene and a hemizygous glucose-6-phosphatedehydrogenase (G6PD) deficiency, diagnosed by an undetectable G6PDactivity in erythrocytes, were found in the patient.

Hb Alperton is the third known mutation at codon 135 of the b-globinchain; Hb Altdorf [b135(H13)Ala→Pro] with increased oxygen affinity (7),and Hb Beckman [b135(H13)Ala→Asp] with a decreased oxygen affinity (8).Hb Beckman was found in a 32-year-old African American woman withchronic anemia, microcytosis and a palpable spleen. While the substitutionof alanine to proline at position 135 in Hb Altdorf results in an unstable Hbvariant with increased affinity for oxygen, substitution of alanine to asparticacid or valine, as in Hb Beckman and Hb Alperton, respectively, has areverse effect. Both Hb Beckman and Hb Alperton showed reduced oxygenaffinity. The site of the amino acid exchange is located within the centralcavity. Other mutations in the central cavity near position 135, such as HbYamagata [b132(H10)Lys→Asn], Hb Hope [b136(H14)Gly→Asp], HbAurora [b139(H17)Asn→Tyr] or Hb Hinsdale [b139(H17)Asn→Lys]showed also decreased oxygen affinity (9).

Only borderline anemia was found in our patient. Other Hb variantswith a strong decrease in oxygen affinity resulted in no or rather mild ane-mia. Clinically, a “grey skin color,” which is related to cyanosis, has beenreported on several occasions (10–12). Even moderate-to-severe anemia inhemoglobinopathies with decreased oxygen affinity like homozygous orheterozygous Hb E [b26(B8)Glu→Lys] are known to be tolerated relativelywell, as these Hbs deliver more oxygen to the tissues. Our patient’s ethnicIndian background did not allow assessment of a possible cyanotic skincolor. However, as cyanosis is only reported from patients with a severeright shift of the oxygen equilibrium curve, we do not expect our patient topresent with cyanosis.

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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