Hemoglobin, 33 (1):54–58, (2009)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260802625733

54

LHEM0363-02691532-432XHemoglobin, Vol. 1, No. 1, November 2008: pp. 1–6Hemoglobin

SHORT COMMUNICATION

A NEW a-GLOBIN VARIANT WITH INCREASED OXYGEN

AFFINITY IN A SWISS FAMILY: Hb FRAUENFELD

[a138(H21)Ser®Phe, TCC>TTC (a2)]

M. Hochuli et al.

Michel Hochuli,1,* Karin Zurbriggen,2,* Marlis Schmid,2 Oliver Speer,2

Philippe Rochat,1 Beat Frauchiger,1 Peter Kleinert,3 Markus Schmugge,2

and Heinz Troxler3

1Department of Internal Medicine, Kantonsspital Frauenfeld, Frauenfeld, Switzerland2Department of Pediatrics, Division of Hematology, University of Zurich, Zurich, Switzerland3Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zurich, Switzerland

� A new a-globin mutation [a138(H21)Ser®Phe] was found in a 55-year-old male probandwith an erythrocytosis known since his youth. Cation exchange high performance liquid chromatog-raphy (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of avariant. The peak area of the variant was approximately one-third that of Hb A suggesting ana-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysisconfirmed the mutation at the protein level. The variant is also detectable with isoelectric focusingand reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138of the a2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser®Pheexchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.

Keywords α-Globin variant, PolyCAT A, High performance liquid chromatography(HPLC), Mass spectrometry (MS), Erythrocytosis

The 55-year-old male patient was hospitalized due to poorly controlledsymptomatic hypertension with headaches and dizziness. The past medical his-tory revealed a mild erythrocytosis known since his youth [maximum hemo-globin (Hb) 18.1 g/dL], treated with repeated, badly tolerated phlebotomies.

Received 1 April 2008; Accepted 1 September 2008.*The first two authors contributed equally to this study.Address correspondence to Dr. Heinz Troxler, Department of Pediatrics, Division of Clinical

Chemistry and Biochemistry, University of Zurich, Steinwiessstrasse 75, CH-8032 Zurich, Switzerland;Tel: +41-44-266-76-40; Fax: +41-44-266-71-69; E-mail: heinz.troxler@kispi.uzh.ch

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nat

iona

l Uni

vers

ity o

f Si

ngap

ore

on 0

6/16

/13

For

pers

onal

use

onl

y.

Hb Frauenfeld [a138(H21)Ser®Phe, TCC>T TC (a2)] 55

Apart from this, the proband was healthy and loved hiking in the mountainareas. Lung function testing and echocardiography did not indicate a pulmo-nary or cardiac cause for the erythrocytosis. The bone marrow examinationwas normal. The patient’s cigarette consumption could hardly accountfor the erythrocytosis (Hb CO 4.6%). Concentrations of methemoglobin(0.0%) in erythrocytes and serum erythropoietin (4.0 U/L; normal range4.0–28.0 U/L) were normal. Red cell indices and blood gas parameters areshown in Tables 1 and 2, respectively.

We decided to screen for a possible hemoglobinopathy with differentwell-established methods. Routine cation exchange high performanceliquid chromatography (HPLC) on a PolyCAT A column (1) and isoelectricfocusing (IEF) (2) of lysate samples were performed as described previ-ously. We applied reversed phase HPLC on a Vydac 124TP54 C4 column(Bucher Biotech, Basel, Switzerland) as described by Kutlar et al. (3) andmatrix-assisted laser desorption ionization-time-of-flight mass spectrometry(MALDI-TOF-MS) (4) for further characterization of the samples. DNA wasprepared from peripheral blood using the QIAamp DNA Blood Mini Kit

TABLE 1 Hematological Parameters of the Heterozygous Carriers of Hb Frauenfeld

ParametersProband

(First DX)Proband

(O2 Study)Brother

(O2 Study)Sister

(O2 Study)

Analysis in: 2000 2008 2008 2008Sex-Age M-50 M-58 M-53 F-55Hb (g/dL) 18.1 17.9 15.4 17.7RBC (1012/L) 5.56 5.64 4.92 5.82PCV (L/L) 0.52 0.51 0.45 0.51MCV (fL) 94.1 90.0 92.0 88.0MCH (pg) 32.6 32.0 31.0 30.0MCHC (g/dL) 34.6 35.2 34.1 34.6Ferritin (μg/L) ND 59.9 33.9 53.7Hb F (%) ND 0.6 0.9 0.7Hb A2 (%) ND 2.6 2.4 2.4Hb A (%) ND 75.4 75.2 74.8Hb Frauenfeld (%) ND 21.4 21.5 22.1

ND, not determined.

TABLE 2 Blood Gas Parameters (Analyzed in 2008)

Parameters Proband Brother Sister

Normal Population

(n = 19)

Heavy Smoker (n = 1)

Light Smoker (n = 1)

p50 (kPa) 3.20 3.30 2.90 4.30 ± 0.2 4.20 4.20Hill coefficient 2.18 2.23 2.35 2.50 ± 0.1 2.53 2.56Hb CO (%) 4.60 5.00 5.60 0.00 5.60 0.70

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nat

iona

l Uni

vers

ity o

f Si

ngap

ore

on 0

6/16

/13

For

pers

onal

use

onl

y.

56 M. Hochuli et al.

(Qiagen, Hombrechtikon, Switzerland). The DNA was amplified using twospecific primer sets for the α1- and α2-globin genes. The α1-globin genewas amplified using the forward primer (5′-AAG TCC ACC CCT TCC TTCCTC ACC-3′) and the reverse primer (5′-TCC ATC CCC TCC TCC CGCCCC TGC CTT TCC-3′). The α2-globin gene was amplified using the sameforward primer as was used for the α1-globin but a different reverse primer(5′-ATG AGA GAA ATG TTC TGG CAC CTG CAC TTG-3′). Hemoglobintonography was performed with 1.5 mL of a heparin blood sample usingthe method described by Duc and Engel (5). Blood gas analysis was performedon a Radiometer NPT7 blood gas analyzer (Radiometer, Copenhagen,Denmark).

In cation exchange chromatography, the Hb variant was observed as anintense signal eluting slightly before Hb A (Figure 1A). In order to confirmthe presence of an Hb variant, the sample was further analyzed withMALDI-TOF-MS (4) (Figure 1B), IEF (Figure 1C), and reversed phase

FIGURE 1 Biochemical analyses of a lysate sample of the Hb Frauenfeld carrier. (A) Cation exchangechromatography reveals a clearly separated peak corresponding to Hb X. (B) The MALDI-TOF massspectrum of the lysate. The mutated α chain is assigned αX. Peaks marked with an asterisk (*) corre-spond to matrix adducts. (C) Isoelectric focusing of the lysate sample. The left lane is a control sample,the right lane is the lysate of the carrier showing the additional band with Hb X.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nat

iona

l Uni

vers

ity o

f Si

ngap

ore

on 0

6/16

/13

For

pers

onal

use

onl

y.

Hb Frauenfeld [a138(H21)Ser®Phe, TCC>T TC (a2)] 57

HPLC (data not shown). In reversed phase HPLC, a distinct signal elutingafter the normal α chain was found. The MALDI-TOF mass spectrumrevealed an additional peak at m/z 15,186 Da with a mass shift of +60 Dacompared to the normal α chain (Figure 1B). The α mutation was finallyidentified by sequencing the α-globin genes, and a C>T transition at thesecond nucleotide of codon 138 of the α2 gene was detected (Figure 2).The TCC>TTC transition at codon 138 on the α2 gene results in a Ser→Pheexchange at position 138 in the α chain. The calculated mass shift of thevariant chain (+60 Da) corresponded well with the observed shift in MS.The new variant was named Hb Frauenfeld for the proband’s place ofresidence.

The patient’s family was also examined (Table 1). The brother andsister turned out to be carriers of the same mutation. The routine bloodtests revealed normal red cell counts for the brother and slightly elevatedHb and RBC values for the patient and his sister. All individuals showedelevated Hb CO values (Table 2) due to cigarette smoking.

Hemoglobin tonography revealed increased oxygen (O2) affinity of thepatient’s Hb. The p50 value for the patient’s whole blood was 3.2 kPa (normal4.3 ± 0.2 kPa; n = 19) and O2 affinity of the other carriers was also increasedto the same extent (Table 2). To evaluate the possible influence of thesmoking habit on the p50 values, we analyzed whole blood of two controls, aheavy smoker with Hb CO comparable to that of the carriers and a lightsmoker with lower Hb CO. We observed normal O2 affinity for both controlsamples (Table 2).

Hb Frauenfeld is the third known mutation at codon 138 of the α-globinchain; the other two are Hb Attleboro [α138(H21)Ser→Pro (TCC>CCC)(α2 or α1)] (6) and Hb Ecuador [α138(H21)Ser→Cys (TCC>TGC (α1)](7). The mutation is located at an internal site of the protein and the

FIGURE 2 DNA sequence (forward) of the carrier of Hb Frauenfeld with the TCC>TTC transition atcodon 138 in the α2 gene. The heterozygous mutation is marked with an arrow.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nat

iona

l Uni

vers

ity o

f Si

ngap

ore

on 0

6/16

/13

For

pers

onal

use

onl

y.

58 M. Hochuli et al.

affected amino acid is at the last position in the H helix. A patient with het-erozygous Hb Attleboro showed a mild anemia with unusually high O2affinity, whereas the hematological parameters of a patient with heterozy-gous Hb Ecuador were normal. Several other mutations in the C-terminalregion near position 138, such as Hb Tokoname [α139(HC1)Lys→Thr (α2or α1], Hb Hanamaki-2 [α139(HC1)Lys→Glu (α2)], Hb Ethiopia[α140(HC2)Tyr→His (α2 or α1)], Hb Legnano [α141(HC3)Arg→Leu (α2or α1], Hb Suresnes [α141(HC3)Arg→His (α2 or α1)], Hb J-Cubujuqui[α141(HC3)Arg→Ser (α2 or α1)], and Hb Nunobiki [α141(HC3)Arg→Cys(α2 or α1)], also showed increased O2 affinity (7).

Impaired O2 release might be a reason why our patient with Hb Frauen-feld was not able to tolerate phlebotomies well. Even with increased O2affinity Hbs, patients may have normal erythropoietin levels that markedlyincrease only after phlebotomy (8). Previous reports have already discussedthe heterogeneity of Hb levels that vary among carriers of the same highaffinity Hb (9), which can explain the normal hematological parameters ofthe brother and the increased Hb values of the patient and his sister (Table 1).

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

REFERENCES

1. Bisse E, Wieland H. High-performance liquid chromatographic separation of human haemoglo-bins. Simultaneous quantitation of foetal and glycated haemoglobins. J Chromatogr. 1988;434(1):95–110.

2. Basset P, Beuzard Y, Garel MC, Rosa J. Isoelectric focusing of human hemoglobin: Its applicationto screening, to the characterization of 70 variants, and to the study of modified fractions ofnormal hemoglobins. Blood. 1978;51(5):971–982.

3. Kutlar F, Kutlar A, Huisman THJ. Separation of normal and abnormal hemoglobin chains byreversed-phase high-performance liquid chromatography. J Chromatogr. 1986;357(1):147–153.

4. Kleinert P, Schmid M, Zurbriggen K, et al. Mass spectrometry: A tool for enhanced detection ofhemoglobin variants. Clin Chem. 2008;54(1)69–76.

5. Duc G, Engel K. A method for determination of oxyhemoglobin dissociation curves at constanttemperature, pH, and PCO2. Respir Physiol. 1969;8(1):118–126.

6. McDonald MJ, Michalski LA, Turci SM, et al. Structural, functional, and subunit assembly proper-ties of Hemoglobin Attleboro [α138(H21)Ser→Pro], a variant possessing a site maturation at acritical C-terminal residue. Biochemistry. 1990;29(1):173–178.

7. Hardison RC, Chui DHK, Giardine B, et al. HbVar: a relational database of human hemoglobinvariants and thalassemia mutations at the globin gene server. Hum Mutat. 2002;19(3):225–233(http://globin.cse.psu.edu).

8. Adamson JW, Finch CA. Hemoglobin function, oxygen affinity, and erythropoietin. Annu RevPhysiol. 1975;37:351–369.

9. Charache S, Achuff S, Winslow R, Adamson J, Chervenick P. Variability of the homeostaticresponse to altered p50. Blood. 1978;52(6):1156–1162.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nat

iona

l Uni

vers

ity o

f Si

ngap

ore

on 0

6/16

/13

For

pers

onal

use

onl

y.