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Hemoglobin
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Hb Hekinan in a Taiwanese Subject: A T Substitution at
Codon 27 of the α1-Globin Gene Abolishes an HaeIII
Site
Hung-Chang Shih ab; Mu-Chin Shih a; Yu-Chang Chang a; Ching-Tien Peng ac;
Tien-Jye Chang b; Jan-Gowth Chang d
a
Department of Laboratory Medicine, China Medical University Hospital, Taichung,
Taiwan
b
Department of Veterinary Medicine, National Chung Hsing University, Taichung,
Taiwan
c
Department of Biotechnology and Bioinformatics, Asia University, Taichung,
Taiwan
d
Department of Laboratory Medicine, Kaohsiung Medical University Hospital,
Kaohsiung, Taiwan
Online Publication Date: 01 October 2007
To cite this Article: Shih, Hung-Chang, Shih, Mu-Chin, Chang, Yu-Chang, Peng, Ching-Tien, Chang, Tien-Jye and
Chang, Jan-Gowth (2007) 'Hb Hekinan in a Taiwanese Subject: A T Substitution at Codon 27 of the α1-Globin Gene
Abolishes an HaeIII Site', Hemoglobin, 31:4, 495 - 498
To link to this article: DOI: 10.1080/03630260701590368
URL: http://dx.doi.org/10.1080/03630260701590368
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Hemoglobin, 31 (4):495–498, (2007)
Copyright © Informa Healthcare USA, Inc.
ISSN: 0363-0269 print/1532-432X online
DOI: 10.1080/03630260701590368
SHORT COMMUNICATION
Hemoglobin, Vol. 31, No. 4, August 2007: pp. 1–6
1532-432X
0363-0269
LHEM
Hemoglobin
Hb HEKINAN IN A TAIWANESE SUBJECT: A G®T SUBSTITUTION AT
CODON 27 OF THE a1-GLOBIN GENE ABOLISHES AN HaeIII SITE
Hung-Chang Shih,1,2 Mu-Chin Shih,1 Yu-Chang Chang,1 Ching-Tien Peng,1,3
Tien-Jye Chang,2 and Jan-Gowth Chang4
1
Department of Laboratory Medicine, China Medical University Hospital, Taichung,
Taiwan
2
Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan
3
Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan
4
Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung,
Taiwan
Hb Hekinan
H.-C.
Shih et in
al.the Taiwanese
䡺
We recently observed a heterozygote for Hb Hekinan in a Taiwanese subject. The molecular
lesion of Hb Hekinan is a substitution of G®T at codon 27 of the a1-globin gene, which abolishes
an HaeIII restriction enzyme site. Hb Hekinan [a27(B8)Glu®Asp, GAG®GAC (a2)] has not
been found in Taiwan. This variant can be detected by high performance liquid chromatography
(HPLC) but not by capillary or cellulose electrophoresis.
Keywords Hb Hekinan, α1-Globin gene, Codon 27, Taiwanese
Hb Hekinan [α27(B8)Glu→Asp, GAG→GAC (α2)] has been reported
in Japanese, Thai, Chinese and Guyanan people (1–4). It is due to a substitution of a glutamic acid by an aspartic acid residue at position 27 of the α1- or
α2-globin chain). We recently observed this variant in the heterozygous state
in a Taiwanese subject. The subject was a 10-years-old boy admitted to the
Department of Pediatric Hematology of China Medical University Hospital
(Taichung, Taiwan) for anemia. The hemogram showed: Hb 9.9 g/dL, RBC
Received 28 February 2007; accepted 6 June 2007.
Address correspondence to Dr. Jan-Gowth Chang, Department of Laboratory Medicine, Kaohsiung
Medical University Hospital, 100 Tzyou 1st Road, Kaohsiung 807, Taiwan; Tel.: +886-7-3115104; Fax:
+886-7-3213931; E-mail: [email protected] or Dr. Tien-Jye Chang, Department of Veterinary
Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 404, Taiwan; Tel.: +886-422840369, Ext. 54; Fax: +886-4-22859222; E-mail: [email protected]
495
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496
H.-C. Shih et al.
4.57 × 1012/L, PCV 0.333 L/L, MCV 72.9 fL, MCH 21.7 pg, MCHC 29.7 g/dL
and ferritin 2.58 ng/mL. Electrophoresis of freshly prepared hemolysates in
cellulose acetate at pH 8.6 or in capillary electrophoresis showed no remarkable change. On electrophoresis by automated high performance liquid
chromatography (HPLC) (PRIMUS CLC 385; Primus Company, Kansas
City, MO, USA), an abnormal peak that was distinguished from Hb A in the
amount of 14.7% was found (Figure 1A). The abnormal hemoglobin (Hb)
could not be detected by capillary electrophoresis (Figure 1B).
The α1-, α2- and β-globin gene analyses were performed and DNA was
isolated from white blood cells using standard methods. The α1-globin
gene was specifically amplified with primers P1 (forward primer, 5′ non
coding area): 5′-CTC TTC TGG TCC CCA CAG AC-3′ and P2 (reverse
primer, 3′ non coding area): 5′-AGG GGC AAG AAG CAT GGC CA-3′, to
amplify the whole coding region and two introns. The α2-globin gene was
specifically amplified with primers P1 and P3 (reverse primer, 3′ non coding area): 5′-CAG GAA GGG CCG GTG CAA GGA G-3′, to amplify the
whole coding region and two introns. The β-globin genes were amplified
with primers P4 (forward primer, 5′ non coding area): 5′-GCT TAC CAA
GCT GTG ATT CC-3′, and P5 (reverse primer, 3′ non coding area): 5′-GGA
CTT AGG GAA CAA AGG AAC C-3′, to amplify the whole coding region
and two introns. The polymerase chain reaction (PCR) conditions were as
follows: the amplification was performed in 50 μL which consisted of 500 ng
genomic DNA, 50 ng each of the primers (P1 + P2 or P1 + P3 or P4 + P5),
0.3% DMSO, 50 μM of each dNTP, 1 × PCR buffer and 2.5 units of Taq
(A) HPLC
(B) CE
HBA 82.8%
HBA 97.7%
HBX 14.7%
HBA2 2.5%
HBA2 2.3%
FIGURE 1 A) An abnormal peak was distinguished from Hb A by HPLC and amounted to 14.7% of the
total Hb. B) The abnormal hemoglobin could not be detected by capillary electrophoresis.
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497
Hb Hekinan in the Taiwanese
polymerase (Perkin Elmer Corporation, Norwalk, CT, USA), using 35 cycles
of 2 min. at 94°C for denaturation, 2 min. at 60°C for annealing, and 3 min.
at 72°C for extension, and a final extension of 5 min. at 72°C, using a
Perkin Elmer Cetus PCR thermocycler. The PCR products were isolated
and sequenced as described previously (5,6). In addition to primers P1, P2,
P3, P4, and P5, the sequencing primers for the α-globin gene were as follows: P6 (reverse primer, intron 1): 5′-CAG GAC GGT TGA GGG TGG
CCT-3′, P7 (forward primer, intron 1): 5′-ACC CCA CCC CTC ACT CGC
TT-3′, P8 (reverse primer, intron 2): 5′-TGC GAG GAA GGC GCC ATC TC3′ and P9 (forward primer, intron 2): 5′-GCA GAG GAT CAC GCG GGT
TG-3′. The sequencing primers for the β-globin genes were: P10 (reverse
primer, intron 1): 5′-GGC AGA GAG AGT CAG TGC CTA-3′, P11 (reverse
primer, exon 2, near intron 2): 5′-CCT GAA GTT CTC AGG ATC CA-3′ and
P12: (forward primer, intron 2): 5′-TGC TAA TCA TGT TCA TAC CT-3′.
The results showed a G→T substitution at the third base of codon 27 of
α1-globin gene (GAG→GAT) that resulted in the substitution of a glutamic
acid for an aspartic acid residue (Figure 2A). This mutation abolishes an
HaeIII restriction enzyme site. We further amplified the mutation area
using primers (P1 + P6), and the PCR products were digested with HaeIII.
The results are shown in Figure 2B.
Hb Hekinan co-migrates with Hb A on cellulose acetate electrophoresis
and capillary electrophoresis, and is difficult to distinguish from Hb A by
(A)
(B)
Normal
200bp
121bp
Codon 27
100bp
73bp
48bp
65bp
0
N
7
73
48
65
204
18
65
204
18
0
M
121
12
HaeIII
FIGURE 2 The results of direct sequencing of the α1-globin gene showed a G→T substitution at codon 27.
Upper case: normal control; lower case: the variant. The results of restriction enzyme HaeIII digestion of
PCR products showed that the patient (lane 3) and positive control (lane 1) had fragments of 121 and
65 bp, respectively (the18 bp fragment was not visible), and the normal control (lane 2) had fragments
of 73, 65 and 48 bp, respectively (the 18 bp fragment was not visualized). M: 100 bp ladder.
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498
H.-C. Shih et al.
these methods. The HPLC procedure using a weak cation exchange material with polyaspartic acid can differentiate these two Hbs. The Hb variant
can be confirmed by either sequencing analysis of the α1-globin gene product
or HaeIII digestion of the amplified α1-globin gene product.
ACKNOWLEDGMENTS
This study was supported by a grant from China Medical University Hospital, Taiwan (DMR-95-0106).
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