SHORT COMMUNICATION

Hb COLIMA [b49(CD8)Ser!Cys]: A NEWHEMOGLOBIN VARIANT

Jose G. Cobian, Maria-Teresa Magana, Francisco J. Perea, and

Bertha Ibarra*

Division de Genetica, Centro de Investigacion Biomedica de Occidente,

Instituto Mexicano del Seguro Social, Sierra Mojada No. 800,

Colonia Independencia, CP 44340, Guadalajara, Jalisco, Mexico

Hb Colima was found in a 52-year-old Mexican Mestizo female, who was born in

Colima, Mexico. This variant was observed during a population screening for the

main hemoglobinopathies in the state of Colima, performed at the Centro de

Investigacion Biomedica de Occidente, Instituto Mexicano del Seguro Social,

Guadalajara, Mexico. The hematological data of the proband, obtained by an

electronic ABX-VEGA counter, were as follows: RBC 4.2961012=L,

Hb 13.6 g=dL, MCV 96.9 fL, MCH 32.0 pg, RDW 13.3%, and the reticulocyte

count was normal. Biochemical analysis was done by conventional methods:[1]

Hb F 1.1%, Hb A2 3.4%, and Hb X 29.3%, that migrated faster than Hb A and

Hb Fannin-Lubbock on cellulose acetate electrophoresis at pH 8.6. Hb X was

quantified by DEAE cellulose chromatography of the stored hemolysate (at �15�C

for two weeks). The isopropanol and butanol stability tests were normal.

Isoelectrofocusing (IEF) was performed on a PhastSystem using PhastGel

(Amersham Biosciences, Piscataway, NJ, USA) IEF media 5–8, and showed an

X band that focused towards the anode from Hb A.

DNA was extracted from the buffy coat by the salting-out method.[2] Direct

DNA sequencing of the b-globin gene showed a C!G substitution at codon 49

(Fig. 1), that results in the replacement of the serine residue by a cysteine in the

b-globin chain. The entire b-globin gene was sequenced by ABI PRISMTM

BigDye Terminator chemistry on an Applied BioSystems 3100 sequencer

(Applied BioSystems, Foster City, CA, USA). The heterozygosity for the C!G

*Corresponding author: Fax: þ52-33-36-18-17-56; E-mail: bibarra@mail.udg.mx

393

DOI: 10.1081=HEM-120016377 0363-0269 (Print); 1532-432X (Online)

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

HEMOGLOBIN

Vol. 26, No. 4, pp. 393–395, 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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substitution was observed in the fragment amplified by the forward primer

(50-ATGGTGCACCTGACTCCTGAGG-30) and the reverse primer (50-CATTCGT-

CTGTTTCCCATTCTA-30) that amplify exons 1 and 2. The mutation was

confirmed by the enzyme Bsp1286 I, since it creates a new restriction site. The

cysteine residue is oriented towards the exterior of the tetramer at position CD8.

This is the second mutation at b49(CD8)Ser; the first was Hb Las Palmas

(!Phe),[3] a slightly unstable variant. There are 13 known human Hb variants with

a cysteine replacement, eight of which are in the b-globin chain, and four of these

are known to form polymers: Hb Harrow [b118(GH1)Phe!Cys], Hb Mississippi

[b44(CD3)Ser!Cys], Hb Ta-Li [b83(EF7)Gly!Cys], and Hb Porto Alegre

[b9(A6)Ser!Cys] (http:==globin.cse.psu.edu=cgi-bin=hbvar).[4] Hb Porto Alegre

exhibits 30% of oligomers, and carries an extra thiol group oriented towards

the exterior of the Hb molecule; the b9 mutation induces polymerization by the

interchain disulfide bonds via the extra cysteine, forming mainly a tetramer of

tetramers.[3] Only tetrameric Hb Porto Alegre was found in the fresh hemolysate,

while polymers were found in the stored hemolysate.[5,6]

Fronticelli et al.[7] engineered a recombinant mutant Hb, Hb Prisca

[bS9C;bC93A;bC112G], that assembles in a polymeric form, and in the presence

of strong reducing agents, the polymer reverts to its tetrameric form; the bS9C

substitution is the same as Hb Porto Alegre.[7] On cellulose acetate electrophoresis

at pH 8.6, the fresh and stored hemolysate (at �15�C for two days) of the proband,

showed an X band. However, with the addition of 2-mercaptoethanol to the buffer

and to the stored hemolysate, the electrophoresis showed a decrease of the X band

and an increase of the band corresponding to Hb A, suggesting that the X band is a

polymeric form of Hb Colima; if we consider that serine and cysteine are neutral

and polar amino acids, the tetrameric form of Hb Colima should not separate from

Figure 1. Part of the electropherogram showing the C!G mutation at codon 49 of the b-globin

gene, resulting in the substitution of the amino acid (aa) serine (Ser) by cysteine (Cys).

394 COBIAN 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.

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Hb A. According to this, 29.3% could be the proportion of the polymer of

Hb Colima of total Hb.

Hb Colima is the third new variant found in Mexico and it could be a

Hb variant with higher levels of oligomers than Hb Porto Alegre.

ACKNOWLEDGMENTS

This work was supported by a grant from Sistema de Investigacion Jose Ma.

Morelos, Consejo Nacional de Ciencia y Tecnologia-REGIONAL No. 19990302015.

REFERENCES

1. Huisman, T.H.J.; Jonxis, J.H.P. The Hemoglobinopathies Techniques of Identification;

Clinical and Biochemical Analysis; Marcel Dekker, Inc.: New York, NY, USA, 1977;

Vol. 6.

2. Miller, S.A.; Dykes, D.D.; Polesky, H.F. A Simple Salting Out Procedure For

Extracting DNA From Human Nucleated Cells. Nucleic Acids Res. 1998, 16,

1215–1216.

3. Malcorra-Azpiazu, J.J.; Balda-Aguirre, M.I.; Diaz-Chico, J.C.; Hu, H.; Wilson, J.B.;

Webber, B.B.; Kutlar, F.; Kutlar, A.; Huisman, T.H.J. Hb Las Palmas or

a2b249(CD8)Ser!Phe, a Mildly Unstable Hemoglobin Variant. Hemoglobin 1988,

12 (2), 163–170.

4. Baudin-Creuza, V.; Fablet, C.; Zal, F.; Green, B.N.; Prome, D.; Marden, M.C.; Pagnier,

J.; Wajcman, H.: Hemoglobin Porto Alegre Forms a Tetramer of Tetramers

Superstructure. Protein Sci. 2002, 11, 129–136.

5. Tondo, C.V.: Study of the Polymerization of Hemoglobin Porto Alegre Tetramers

Disulfide Bridge Formation. An. Acad. Brasil. Cienc. 1971, 43, 337–348.

6. Tondo, C.V.: Hemoglobin Porto Alegre: An Asymmetric Tetramer With Only One

Abnormal b Chain in the Aged Hemolysate of Erythrocytes From the Heterozygous

Carrier (a2AbAbSer!Cys). An. Acad. Brasil. Cienc. 1972, 44, 337–348.

7. Fronticelli, C.; Arosio, D.; Bobofchak, K.M.; and Vasquez, G.B. Molecular

Engineering of a Polymer of Tetrameric Hemoglobins. Proteins 2001, 44, 212–222.

Received May 29, 2002

Accepted June 24, 2002

Hb COLIMA [b49(CD8)Ser!Cys] 395

©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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