SHORT COMMUNICATION

A NOVEL b-GLOBIN VARIANT: Hb POCOS DECALDAS [b61(E5)Lys!Gln]

Elza M. Kimura,1 Simone B. Jorge,1 Satie H. Ogo,3

Maristela Cesquini,3 Dulcineia M. Albuquerque,2

Andre Fattori,2 Sara T. O. Saad,2 Fernando F. Costa,2

and Maria F. Sonati1,*

1Department of Clinical Pathology and 2Department of Clinical

Medicine, School of Medical Sciences and 3Department of

Biochemistry, Institute of Biological Sciences,

State University of Campinas, UNICAMP, P.O. Box 6111,

Campinas (SP), Brazil 13083-970

A novel hemoglobin (Hb) variant was found during a screening program for

hemoglobinopathies in blood donors at the Hematology Center of the State

University of Campinas, Campinas, Brazil. The carrier was a 30-year-old

Caucasian woman of Native Indian and Italian origin, born in Pocos de Caldas,

a city in the State of Minas Gerais, Southeastern Brazil. The new Hb was

distinguished as an abnormal band, faster than Hb A, at alkaline pH (Fig. 1a), that

moved like Hb A at acid pH.[1] Isoelectrofocusing (IEF) with an REP Hemoglobin

IEF Kit (Helena Laboratories, Beaumont, TX, USA), at pH ranges varying from

6.0 to 8.0, showed a fast band moving to the anode (Fig. 1b). Urea Triton X-100

polyacrylamide gel electrophoresis[2] showed the presence of a b chain variant that

was barely discernible from the normal b chain (Fig. 1c). Stability tests[1] were

normal. The Hb oxygen equilibria were performed by the method of Rossi-Fanelli

and Antonini,[3] in stripped Hb and in the presence of organic polyphosphate at a

pH between 7.0 and 8.0. Corroborating the clinical manifestation, Hb Pocos de

Caldas showed functional properties similar to that of normal Hb.

Molecular analyses involved the amplification of a 661 bp fragment of the

b-globin gene, using primers P1 (50-GCCAAGGACAGGTACGGCTGTCATC-30)

*Corresponding author. Fax: þ(55-19) 3788-9434; E-mail: sonati@fcm.unicamp.br

385

DOI: 10.1081=HEM-120016375 0363-0269 (Print); 1532-432X (Online)

Copyright # 2002 by Marcel Dekker, Inc. www.dekker.com

HEMOGLOBIN

Vol. 26, No. 4, pp. 385–388, 2002

©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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Figure 1. a) Hb electrophoresis on cellulose acetate at pH 8.9. Lane 1: normal control; lane 2:

proband; lane 3: proband’s mother; lane 4: proband’s brother; lane 5: proband’s sister. b) IEF showing

Hb Pocos de Caldas in relation to Hbs A, F, and S (pH range: 6.0–8.0). Lane 1: AS control; lane 2:

AF control; lanes 3–6: proband and proband’s mother, brother, and sister, respectively; lane 7: AA

control. c) Polyacrylamide gel electrophoresis in the presence of Urea Triton X-100 of the globin

chains. Top: anode; bottom: cathode. P: proband; M: proband’s mother; N: newborn normal control.

d) b-Globin gene sequencing showing the replacement AAG!CAG as the molecular basis of Hb

Pocos de Caldas.

a)

b)

c)

386 KIMURA ET AL.

©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

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and P5 (50-CCCTTCTTCCTATGACATGAACTTAACCAT-30) (spanning from

position �140, relative to the Cap site, to position þ521, at the beginning of

IVS-II).[4] The amplification reactions were carried out in a 50mL volume

containing 500 ng of genomic DNA, 50 ng of each primer (P1þP5), 50 mM of

each dNTP, 1X polymerase chain reaction (PCR) buffer (Taq polymerase buffer),

and 2.5 units of Taq DNA polymerase (Amersham Pharmacia Biotech,

Piscataway, NJ, USA). The PCR conditions were as follows: initial denaturation

for 2 mins at 94�C, followed by 35 cycles of 30 seconds at 94�C for denaturation,

45 seconds at 64�C for annealing, and 1 min at 72�C for extension, and a final

extension of 7 mins at 72�C, using a Perkin Elmer Cetus (Norwalk, CT, USA) PCR

thermocycler. Direct sequencing of the PCR products was performed by using the

Big DyeTM Terminator Cycle Sequencing Kit and the ABI PRISMTM 377

Genetic Analyzer (PE Applied BioSystems, Foster City, CA, USA), with primer

P2 (50-TTTGCTTCTGACACAACTGT-30).[4] A base substitution was detected at

codon 61 (AAG!CAG), causing the replacement Lys!Gln in the corresponding

position of the b chain (Fig. 1d). This mutation was confirmed by sequencing the

Figure 1. Continued.

Table 1. Hematological Data of the Hb Pocos de Caldas (PC) Carrier and Her Family

Parameters Proband Mother Brother Sister

RBC (1012=L) 4.65 4.26 5.41 3.98

Hb (g=dL) 14.4 13.3 15.5 11.9

PCV (L=L) 0.451 0.403 0.477 0.367

MCV (fL) 97.0 95.0 87.0 92.0

MCH (pg) 30.9 31.3 28.7 29.8

RDW (%) 15.0 15.5 18.4 15.5

Electrophoretic

profile

A2þAþ PC A2þAþ PC A2þAþ PC A2þAþ PC

Hb A2 (%) 2.37 2.60 2.25 2.34

Hb F (%) 0.66 0.90 0.76 0.45

d)

Hb POCOS DE CALDAS [b61(E5)Lys!Gln] 387

©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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DNA opposite strand and familial analysis, which revealed that the carrier’s

mother, brother, and sister also have the same b-globin alteration (see Table 1).

This is the fourth description of a mutation at b61(E5)Lys: Hb N-Seattle

(!Glu) was found in a Black American blood donor,[5] Hb Hikari (!Asn)

detected in a Japanese family[6] and Hb Bologna (!Met) encountered in a North

Italian family.[7] The mutations found at this position (external contacts of the Hb

molecule) do not cause clinical manifestations, since all the carriers described so

far have been asymptomatic.

ACKNOWLEDGMENTS

Funding came from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo

(FAPESP) (grant 1997=11725-1) and Conselho Nacional de Desenvolvimento Cientıfico

e Tecnologico (CNPq), Brazil.

REFERENCES

1. Dacie, J.V.; Lewis, S.M. Practical Haematology, 8th Ed; Churchill Livingstone:

London, England, 1995.

2. Alter, B.P.; Goff, S.C.; Efremov, G.D.; Gravely, M.E.; Huisman, T.H.J. Globin Chain

Electrophoresis: A New Approach to the Determination of the Gg=Ag Ratio in Fetal

haemoglobin and to Studies of the Globin Synthesis. Br. J. Haematol. 1980, 44,

527–534.

3. Rossi-Fanelli, A.; Antonini, E. Studies on the Oxygen and Carbon-Monoxide

Equilibria of Human Myoglobin. Arch. Biochem. Physiol. 1958, 77, 428–492.

4. Miranda, S.R.P.; Fonseca, S.F.; Figueiredo, M.S.; Yamamoto, M.; Grotto, H.Z.W.;

Saad, S.T.O.; Costa, F.F. Hb Koln [a2b2 98 (FG5) Val!Met] Identified by DNA

Analysis in a Brazilian Family. Braz. J. Genet. 1997, 20, 745–748.

5. Jones, R.T.; Brimhall, B.; Huehns, E.R.; Motulsky, A.G. Structural Characterization

of Hemoglobin N Seattle: a2Ab2 61 Lys!Glu. Biochim. Biophys. Acta 1968, 154,

278–283.

6. Nakatsuji, T.; Miwa, S.; Hattori, Y.; Ohba, Y.; Miyaji, T.; Miyata, H.; Shinohara, T.;

Matsui, Y. A Further Example of Hemoglobin Hikari (b61[E5]Lys!Asn).

Hemoglobin 1981, 5 (5), 487–492.

7. Marinucci, M.; Giuliani, A.; Maffi, D.; Massa, A.; Giampaolo, A.; Mavilio, F.;

Zannotti, M.; Tentori, L. Hemoglobin Bologna (a2b2 61 (E5) Lys!Met) an

Abnormal Human Hemoglobin With Low Oxygen Affinity. Biochim. Biophys. Acta

1981, 668, 209–215.

Received May 24, 2002

Accepted July 4, 2002

388 KIMURA ET AL.

©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

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