Hb Tunis [beta124(H2)Pro>Ser] was reported from Tunisia in 1988. This hemoglobin variant was detected by isoelectric focusing moving just ahead of Hb A. It cannot be identified by standard hemoglobin electrophoresis due to its similar mobility to Hb A. It has normal stability and oxygen affinity and does not produce any clinical symptoms. Here, we report a heterozygous Hb Tunis [beta124(H2)Pro>Ser] case discovered for the first time in Turkey in a premarital screening program. This hemoglobin variant can be identified with high performance liquid chromatography analysis confirmed with DNA sequencing. We emphasize in our study the importance of an interdisciplinary collaborative study at the provincial basis for the success of the hemoglobinopathy control program

Atalay, Ayfer

Atalay, Erol Ömer

Bahadır, Anzel

Demirtepe, Sanem

Koyuncu, Hasan

Köseler, Aylin

Öztürk, Onur

Turkish Journal of Hematology

Pamukkale Üniversitesi Açık Erişim Arşivi

First observation of Hb Tunis [beta124(H2)
Pro>Ser] in Turkey
Türkiye’de gözlenen ilk Hb Tunis [beta124(H2)Pro>Ser] olgusu
Aylin Köseler1, Hasan Koyuncu2, Onur Öztürk1, Anzel Bahadır1, Sanem Demirtepe1, Ayfer Atalay1, 
Erol Ömer Atalay1
1Department of Biophysics, Pamukkale University Faculty of Medicine, Denizli, Turkey
2Turkish Ministry of Health Hemoglobinopathy Center, Denizli, Turkey
Address for Correspondence: Prof. Erol Ömer Atalay, Pamukkale Üniversitesi Tıp Fakültesi Biyofizik Anabilim Dalı (Morfoloji) 20200 Denizli, Turkey
 Phone: +90 258 211 90 27 Office: +90 258 296 24 87 E-mail: eatalay@pau.edu.tr
doi:10.5152/tjh.2010.11
Abstract
Hb Tunis [beta124(H2)Pro>Ser] was reported from Tunisia in 1988. This hemoglobin variant was detected by isoelectric 
focusing moving just ahead of Hb A. It cannot be identified by standard hemoglobin electrophoresis due to its similar 
mobility to Hb A. It has normal stability and oxygen affinity and does not produce any clinical symptoms. Here, we report 
a heterozygous Hb Tunis [beta124(H2)Pro>Ser] case discovered for the first time in Turkey in a premarital screening pro-
gram. This hemoglobin variant can be identified with high performance liquid chromatography analysis confirmed with 
DNA sequencing. We emphasize in our study the importance of an interdisciplinary collaborative study at the provincial 
basis for the success of the hemoglobinopathy control program. (Turk J Hematol 2010; 27: 120-2)
Key words: Hemoglobinopathy, abnormal hemoglobin, Hb Tunis
Received: September 12, 2008  Accepted: April 8, 2009
Özet
Hb Tunis [beta124(H2)Pro>Ser] ilk kez 1988 yılında Tunus’da bildirilmiştir. Bu hemoglobin varyantı alkali ve asit ortamda 
yapılan standart elektroforez yöntemleri ile ayrılamamaktadır. İzoelektrik odaklama elektroforezinde (IEF-isoelectric focus-
ing) ise Hb A’dan biraz daha hızlı bir davranış ortaya koymaktadır. Hb Tunis normal stabilite ve normal oksijen afinitesine 
sahip olup herhangi bir klinik belirti vermemektedir. Premarital tarama çalışmasında saptanan heterozigot Hb Tunis olgu-
muz, Türkiye ilk kez bildirilmektedir. Bu hemoglobin varyantı HPLC yöntemi ile ayrılabilmiş ve DNA dizi analizi ile 
doğrulanmıştır. Bu çalışmamızda ayrıca, hemoglobinopati kontrol programlarının başarılı biçimde uygulanabilmesi için yöre-
sel düzeyde disiplinler arası işbirliğinin önemini vurgulamaktayız. (Turk J Hematol 2010; 27: 120-2)
Anahtar kelimeler: Hemoglobinopati, anormal hemoglobin, Hb Tunis
Geliş tarihi: 12 Eylül 2008  Kabul tarihi: 8 Nisan 2009
120 Case Report
Introduction
Many hemoglobin variants other than Hb S have been 
reported from Turkey [1,2]. In the Denizli province of Turkey, 
the most frequent hemoglobin variants are Hb D-Los Angeles 
and Hb G-Coushatta [3]. In addition to the reviewed hemoglo-
bin variants in Turkey, Hb D-Ouled Rabah, Hb Yaizu and Hb 
Beograd have also been reported from the Denizli province 
[4-6]. Hb Tunis [beta124(H2)Pro>Ser] was reported from 
Tunisia by Mrad et al. in 1988 [7]. This hemoglobin variant was 
detected by isoelectric focusing (IEF) moving just ahead of Hb 
A. It has normal stability and oxygen affinity and does not 
cause any clinical symptoms. It is also not detectable by con-
ventional hemoglobin electrophoresis. The Hemoglobinopathy 
Control Program has been applied in our province since 2005 
in collaboration with the Turkish Ministry of Health Denizli 
Hemoglobinopathy Center and Pamukkale University Medical 
Faculty, leading to prenatal diagnosis in the cases of pregnan-
cies at risk. We report herein the first observation of Hb Tunis 
[beta124(H2)Pro>Ser] hemoglobin variant in Turkey from the 
Denizli province determined during the premarital screening 
program. 
Case Report 
We report in this study identification of the heterozygous 
Hb Tunis [beta124(H2)Pro>Ser] in a 21-year-old male living in 
the Denizli province, located in the Aegean region of Turkey. 
Written informed consent was obtained for the laboratory tests 
and DNA analysis from the proband, and the sample was 
deposited at the DNA Bank of the Department of Biophysics 
as an anonymous sample. Hemoglobin electrophoresis at 
alkaline and acid pH, DE-52 column chromatography, and 
non-radioactive fluorescence-based DNA sequencing as previ-
ously published were performed [8]. For the sequencing of the 
beta globin gene exon 3, the forward primer PAM600 (5’-CAA 
TGT ATC ATG CCT CTT TGC ACC-3’) and reverse primer 
PAM603 (5’-CAC TGA CCT CCC ACA TTC CC-3’) were used. 
High performance liquid chromatography (HPLC) was obtained 
with BioRad Variant II system, USA. Blood cell counts were 
done with Beckman Coulter AcT10 instrument, USA. 
The hemoglobin variant could not be identified by alkaline 
or acid pH in agarose based electrophoresis and behaved like 
Hb A. Results of analysis of the red blood cell parameters were 
as follows: Hb 15.6 g/dl, RBC 5.50 106/mm3, Hct 47.7%, MCV 
87.0 fL, MCH 28.3 pg, MCHC 32.7 g/dl, and RDW 12.6%. In 
DE-52 microcolumn chromatography, Hb A2 was eluted (3.1%) 
but Hb X could not be eluted with Hb S buffer (0.2 M glycine / 
0.014 M NaCl / 0.01 % KCN). Changing buffer content in the 
NaCl concentration does contribute to the elution of Hb X. The 
Hb variant produces a double peak at the Hb A window slightly 
slower than Hb A in HPLC analysis, as shown in Figure 1. 
According to the HPLC results, the Hb ratios for Hb A0, Hb X, 
Hb A2 and Hb A1c were 45.71%, 48.84%, 3.09% and 2.36%, 
respectively. DNA sequencing revealed the hemoglobin variant 
as a mutation at beta globin gene exon 3 codon 124 
(CCA>TCA, Pro>Ser), as shown in Figure 2.
Discussion
In this study, Hb Tunis was identified by DNA sequence 
analysis, which confirmed the C-to-T mutation at the beta glo-
bin gene codon 124. Hb Tunis was reported once in the world 
populations by Mrad et al. [7] from Tunisia. To the best of our 
knowledge, our case is the second observation in the world 
population and is the first reported case in Turkey. This muta-
tion causes the replacement of proline residue into serine. 
Proline has no ionizable groups, and all conformations like cis- 
and trans- are almost isoenergetic. When proline is in the 
protein structure, it cannot donate a hydrogen bond for stabi-
lizing the protein structure. On the other hand, serine is a neu-
tral polar molecule that has a tendency to remain on the sur-
face of the proteins. The replacement proline into serine at beta 
globin codon 124 does not change its standard electropho-
retic characteristics at alkaline and acid medium. Only IEF 
electrophoresis can detect Hb Tunis [beta124(H2)Pro>Ser] as 
a slightly faster moving band compared to Hb A [7] due to the 
slight difference in the isoelectric points of the proline and ser-
ine residues. The presence of serine also changes the chro-
matographic behavior of the hemoglobin molecule slightly. This 
Köseler et al.
Hb Tunis case in TurkeyTurk J Hematol 2010; 27: 120-2 121
Figure 1. HPLC result of heterozygous Hb Tunis case
Hd Tunis H
dA
0
H
dA
2
H
dA
1c
mAU
25
5
15
20
0
0 2 4 6 8 min
10
Figure 2. DNA sequencing of hemoglobin AX (beta 124, CCA>TCA, Y=C/T)
Codon 124
70
AC C C CY C C CA A AG GT
change cannot be observed in DE-52 microcolumn chroma-
tography, but it can be detected by the HPLC system, which 
has resolution capacity. In the HPLC system, we observed a 
slightly slower peak at the Hb A window. This behavior is due 
to the weak interaction of the serine residue with the HPLC 
column matrix. In the HPLC system, the parameters should be 
standardized very carefully. If the experimental HPLC analysis 
parameters are not controlled and standardized, such hemo-
globin variants can be bypassed easily.
Beta globin gene codon 124 resides in the external region 
of the hemoglobin molecule and is located in the α1β1 contact 
region of the hemoglobin tetramer. During transition of the 
hemoglobin molecule from oxy-form to deoxy-form, the relative 
displacement of the β-chain to the α-chain is greater at the 
α1β2-contact than at the α1β1-contact [9]. Therefore, it is 
expected that the effect of the mutation on the structure and 
function the hemoglobin molecule is absent or very small and 
thus does not manifest in clinical symptoms. This heterozy-
gous Hb Tunis case is an apparently healthy male. The place 
of the codon 124 with its characteristic features confirms this 
issue. Since homozygous Hb Tunis and other combinations 
with alpha and beta thalassemia are not yet reported, the 
clinical significance of this mutation remains unclear in such 
conditions.
Concerning the historical background, North African popu-
lations were affected by different Mediterranean populations 
due to the population movements. The Turkish influence was 
particularly notable in Tunisia and Algeria during the 14th-19th 
centuries [10]. Although molecular genetic data like beta globin 
gene cluster haplotypes is absent, Hb Tunis could have been 
introduced in the Denizli province gene pool due to these his-
torical population movements.
In premarital screening programs, the nature of any hemo-
globin variant detected by HPLC that is of potential clinical 
relevance should be confirmed by alternative techniques [11]. 
Premarital screening leading to prenatal diagnosis of the 
hemoglobinopathies has been applied in our province since 
2004. Identification of the hemoglobin variants poses problems 
in the premarital control program in Denizli province due to the 
heterogeneous structure of the province. According to the 
results of our registered cases, Hb D-Los Angeles and Hb 
G-Coushatta were observed frequently [3]. Hb Beograd, Hb 
Yaizu and Hb D-Ouled Rabah have also been reported from 
our province [4-6]. We would also like to emphasize the impor-
tance of an interdisciplinary collaborative study at the provincial 
basis to facilitate the success of the hemoglobinopathy control 
program.
In conclusion, we report Hb Tunis [beta124(H2)Pro>Ser] for 
the first time in Turkey from the Denizli province. We emphasize 
that the exact identification of many hemoglobin variants can 
be easily bypassed with the application of limited laboratory 
techniques like standard hemoglobin electrophoresis and even 
with HPLC methods. The standardization of the protocols and 
algorithmic approach used in hemoglobinopathy control pro-
grams is another important issue to be addressed.
Acknowledgements 
The authors are grateful to the staff of the Turkish Ministry 
of Health Denizli Hemoglobinopathy Center and MSc student 
of the Biophysics Department, Çağıl Coşkun, for their valuable 
contributions. This work was supported by the Pamukkale 
University Research Fund Projects (Nos. 2002TPF017, 
2005SBE001, 2005SBE002, 2006SBE004 and 2006TPF001).
Conflict of interest
No author of this paper has a conflict of interest, including 
specific financial interests, relationships, and/or affiliations rel-
evant to the subject matter or materials included in this manu-
script.
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Köseler et al.
Hb Tunis case in Turkey Turk J Hematol 2010; 27: 120-2122


Türkiye’de gözlenen ilk Hb Tunis [beta124(H2)Pro>Ser] olgusu

Hb Tunis [beta124(H2)Pro>Ser] was reported from Tunisia in 1988. This hemoglobin variant was detected by isoelectric focusing moving just ahead of Hb A. It cannot be identified by standard hemoglobin electrophoresis due to its similar mobility to Hb A. It has normal stability and oxygen affinity and does not produce any clinical symptoms. Here, we report a heterozygous Hb Tunis [beta124(H2)Pro>Ser] case discovered for the first time in Turkey in a premarital screening program. This hemoglobin variant can be identified with high performance liquid chromatography analysis confirmed with DNA sequencing. We emphasize in our study the importance of an interdisciplinary collaborative study at the provincial basis for the success of the hemoglobinopathy control program. (Turk J Hematol 2010; 27: 120-2

Koseler, A

Koyuncu, H

Ozturk, O

Bahadir, A

Demirtepe, S

Atalay, A

Atalay, EO

First observation of Hb Tunis [beta124(H2) Pro > Ser] in Turkey

http://acikerisim.pau.edu.tr:8080/xmlui/bitstream/11499/6346/1/TJH_27_2_120_122.pdf

Türkiye’de gözlenen ilk Hb Tunis [beta124(H2)Pro>Ser] olgusu

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