Objective: The aim of this study was to determine the laboratory diagnosis and genetic origins of the hemoglobin (Hb) variants, Hb D-Los Angeles and Hb Beograd observed frequently in our region. Material and Methods: Hb variants were investigated in one Hb D-Los Angeles and two Hb Beograd families. These families were unrelated with each other. For the determination of Hb variants, alkaline/acid electrophoresis, HPLC, DE-52 micro-column chromatography procedures were applied. Mutations were determined by non-radioactive fluorescence automated DNA sequencing. Beta globin gene cluster haplotypes were identified by RFLP analysis at seven loci known as ε-Hinc II, Gγ-Hind III, Aγ-Hind III, 5′ψβ-Hinc II, 3′ψβ-Hinc II, β-Ava II ve 3′β-Hinf I. Results: Three novel beta globin gene cluster haplotypes were identified as in relation with Hb D-Los Angeles [--+-+++], Hb Beograd [+----++ and -+-(+/-)(+/-)+(+/-)]. These haplotypes were reported for the first time in the world population Conclusion: In this study we emphasize the importance of DNA seqeuncing and other laboratory procedures for the identification of Hb variants in premarital diagnosis. On the other hand we discuss also the genetic origins of these Hb variants

Akar, E.

Akar, N.

Atalay, Ayfer

Atalay, E.Ö.

Bahadır, Anzel

Koyuncu, H.

Köseler, Aylin

Pamukkale Üniversitesi Açık Erişim Arşivi

17Research Article  
Hb D-Los Angeles [beta121(GH4)Glu>Gln] and Hb 
Beograd [beta121(GH4)Glu>Val]: Implications for 
their laboratory diagnosis and genetic origins
Hb D-Los Angeles [beta121(GH4)Glu>Gln] ve Hb Beograd [beta121(GH4)Glu>val]: 
laboratuar tanıları ve gensel kökenleri
Anzel Bahadır1, Aylin Köseler1, Ayfer Atalay1, Hasan Koyuncu2, Ece Akar3, Nejat Akar3, 
Erol Ömer Atalay1
1Pamukkale University Medical Faculty Department of Biophysics, Denizli, Turkey
2Turkish Ministry of Health Denizli Hemoglobinopathy Center, Denizli, Turkey
3Ankara University Medical Faculty Department of Pediatric Hematology, Ankara, Turkey
Address for Correspondence: Prof. Dr. Erol Ömer Atalay, Pamukkale Üniversitesi Tıp Fakültesi Biyofizik Anabilim Dalı (Morfoloji) 20020 Kınıklı, Denizli, Türkiye
 Phone: +90 258 211 90 27 - +90 258 295 24 87 E-mail: eatalay@pau.edu.tr
Abstract
Objective: The aim of this study was to determine the laboratory diagnosis and genetic origins of the hemoglobin (Hb) 
variants, Hb D-Los Angeles and Hb Beograd observed frequently in our region.
Material and Methods: Hb variants were investigated in one Hb D-Los Angeles and two Hb Beograd families. These 
families were unrelated with each other. For the determination of Hb variants, alkaline/acid electrophoresis, HPLC, DE-52 
micro-column chromatography procedures were applied. Mutations were determined by non-radioactive fluorescence 
automated DNA sequencing. Beta globin gene cluster haplotypes were identified by RFLP analysis at seven loci known 
as ε-Hinc II, Gγ-Hind III, Aγ-Hind III, 5’ψβ-Hinc II, 3’ψβ-Hinc II, β-Ava II ve 3’β-Hinf I.
Results: Three novel beta globin gene cluster haplotypes were identified as in relation with Hb D-Los Angeles [--+-+++], 
Hb Beograd [+----++  and -+-(+/-)(+/-)+(+/-)]. These haplotypes were reported for the first time in the world population
Conclusion: In this study we emphasize the importance of DNA seqeuncing and other laboratory procedures for the 
identification of Hb variants in premarital diagnosis. On the other hand we discuss also the genetic origins of these Hb 
variants. (Turk J Hematol 2009; 26: 17-20)
Key words: Hb Beograd, Hb D-Los Angeles, laboratory diagnosis, genetic origin, premarital diagnosis
Received: March 25, 2008  Accepted: November 2, 2008
Özet
Amaç: Bu çalışmamızın amacı, yöremizde sıklıkla karşılaşılan Hb D-Los Angeles ve Hb Beograd’ın laboratuar tanısı ve 
gensel kökenlerine ilişkin sonuçların irdelenmesidir.
Yöntem ve Gereçler: Bu çalışmada, aralarında akrabalık ilişkisi olmayan bir HbD-Los Angeles ve iki Hb Belgrad ailesi 
incelenmiştir. Hemoglobin (Hb) varyantlarının tanımlanmasında alkali ve asit hemoglobin elektroforezi, HPLC, DE-52 mik-
rokolon kromatografisi yöntemleri kullanılmıştır. Mutasyonların saptanmasında flüoresan işaretli dizi analiz yöntemi 
uygulanmıştır. Beta globin gen ailesi haplotiplerinin belirlenmesinde ise ε-Hinc II, Gγ-Hind III, Aγ-Hind III, 5’ψβ-Hinc II, 3’ψβ-
Hinc II, β-Ava II ve 3’β-Hinf I olmak üzere toplam yedi odakta RFLP analizi yapılmıştır.
Introduction
Premarital screening is one of the most important compo-
nents in the hemoglobinopathy control programs. The correct 
identification of the mutations elucidates the success of the 
prenatal diagnostic procedures to be applied in a timely and 
cost-effective manner in the case of pregnancy. The premarital 
molecular diagnosis is also helpful in the reduction of the stress 
on the families under interest regarding the prenatal diagnostic 
approaches. Since the first chorionic villus sampling (CVS)-
based reported case in 1990 [1], prenatal diagnosis has been 
available in different centers in Turkey like Hacettepe University 
(Ankara), Boğaziçi University (Istanbul), and Çukurova University 
(Adana) [2]. In our province, prenatal diagnosis has been 
applied since 2004. The premarital screening and counseling in 
genetics has been applied by law since 1998 in the centers 
established by the Turkish Ministry of Health in Hatay, Adana, 
Mersin and other at- risk provinces like Denizli province [2,3]. 
Hb Beograd [β121(GH4)Glu→Val, GAA→GTA] is a variant with 
normal clinical presentation in heterozygous carriers. The 
homozygous form of Hb Beograd is not known. It has been 
reported from Yugoslavia, Turkey, Australia and New Zealand 
[4]. On the other hand, Hb-Los Angeles (also known as 
D-Punjab, Hb North Carolina, D-Portugal, D-Chicago, and Oak 
Ridge) is an abnormal Hb at codon 121 of the beta globin gene 
resulting in the amino acid substitution of glutamine for glu-
tamic acid. Hb D-Los Angeles is the most frequent abnormal 
hemoglobin observed in Turkey [5], especially in the Denizli 
province [3]. In premarital screening programs, these abnormal 
Hbs are easily confused with each other and also with other 
D-like Hbs like Hb G-Coushatta, due to their similar electro-
phoretic and chromatographic behavior. Since these abnormal 
hemoglobins reside on the same codon, they are also mixed 
with procedures like the Eco RI restriction enzyme protocols at 
the gene level. In this study, we aimed to discuss the labora-
tory diagnosis of Hb Beograd and Hb D-Los Angeles as well 
as their genetic origins according to the beta globin gene clus-
ter haplotypes.
Materials and Methods
We analyzed three individuals from one Hb Beograd family, 
one unrelated heterozygous Hb Beograd and one unrelated 
homozygous Hb D-Los Angeles cases. These cases were from 
three unrelated families. All cases were detected during a pre-
marital screening program. Blood samples were collected in 
EDTA vacutainers and DNA was extracted from peripheral 
blood using the standard phenol-chloroform procedure. Written 
informed consent was obtained from these individuals for DNA 
analysis and the samples were deposited into our Department’s 
DNA Bank as anonymous samples. Alkaline and acid Hb elec-
trophoresis, DE-52 micro column chromatography and non 
radioactive DNA sequencing were done as previously reported 
[2]. High performance liquid chromatography (HPLC) results 
were obtained with BioRad Variant II system. Beta globin gene 
cluster haplotype analyses were performed as previously 
reported elsewhere [6].
Results
Hb D-Los Angeles [β121(GH4) Glu→Gln] and Hb Beograd 
[β121(GH4) Glu→Val] have the same electrophoretic mobility on 
alkaline and acid Hb electrophoresis. Both behave like Hb S in 
alkaline pH and present Hb A-like mobility in acidic pH. In HPLC 
analysis, Hb Beograd [β121(GH4) Glu→Val] can be separated 
with a retention time of 4.19 min (Figure 1). Beta globin gene 
cluster haplotypes of the cases are shown in Table 1. In the 
Bulgular: Bu çalışmada, Hb D-Los Angeles ile ilgili olarak bir tane [--+-+++], Hb Beograd için ise iki tane [+----++] ve 
[-+-(+/-)(+/-)+(+/-)] yeni beta globin haplotipi saptanmıştır. Bu haplotipler literatürde ilk kez bildirilmektedir.
Sonuç: Bu çalışmada, özellikle premarital tanıda kullanılan yöntemler ile DNA dizi analizinin önemi vurgulanmaktadır. Diğer 
taraftan bu hemoglobin türlerinin gensel kökenlerine ilişkin veriler irdelenmektedir. (Turk J Hematol 2009; 26: 17-20) 
Anahtar kelimeler: Hb Beograd, Hb D-Los Angeles, laboratuvar tanısı, gensel köken, premarital tanı
Geliş tarihi: 25 Mart 2008 Kabul tarihi: 02 Kasım 2008
Bahadır et al.
Hb Beograd and Hb D-Los Angeles: Laboratory diagnosis and genetic origins Turk J Hematol 2009; 26: 17-2018
Table 1. Beta globin gene cluster haplotypes of the unrelated Hb D-Los Angeles cases and Hb Beograd family 
  Hemoglobin  5’-ε Gγ Aγ 5’-ψβ 3’-ψβ 5’-β 3’-β
    Hinc II Hind III Hind III Hinc II Hinc II Ava II Hinf I 
Case #1 Hb D-Los Angeles (homozygous) -/- -/- +/+ -/- +/+ +/+ +/+
Case #2 Hb Beograd (heterozygous) -/- +/+ -/- +/- +/- +/+ +/-
Hb Beograd Family (*)
Case #3 Hb Beograd (heterozygous) +/+ -/- -/- -/- -/- +/- +/+
Case #4 Normal  +/- +/- +/- +/- +/- +/+ +/+
Case #5 Hb Beograd (heterozygous) +/- +/- +/- +/- +/- +/+ +/+
Associated Haplotype  + - - - - + +
(*) Case #3 propositus, case #4 mother and case #5 sister for the Hb Beograd family
case of Hb Beograd, there were two different haplotypes, as 
[+----++] and [-+-(+/-) (+/-) + (+/-)], in two unrelated families. For 
the homozygous Hb D-Los Angeles case, the related haplotype 
is a novel haplotype, as [--+-+++]. 
Discussion
Since the retention times of Hb D-Los Angeles [β121(GH4) 
Glu→Gln] and Hb Beograd [β121(GH4) Glu→Val] are almost 
similar as observed in D-window, they cannot be differentiated 
easily in premarital screening using HPLC. Laboratory identifi-
cation of the Hb D-Los Angeles and Hb Beograd is not pos-
sible with electrophoretic and chromatographic techniques in 
premarital screening programs. As far as molecular identifica-
tion is concerned, Eco RI restriction enzyme digestion protocol 
is also not sufficient. Since EcoRI digestion cannot differentiate 
Hb Beograd from Hb D-Los Angeles, as the mutation lies in the 
same codon, EcoRI/Tsp509I dual restriction enzyme digestion 
can be used to differentiate these variants. The restriction 
enzyme digestion protocol is convenient, rapid and feasible, 
especially in populations where these mutations are prevalent 
[7]. DNA sequencing is an important issue for the application of 
the exact determination of the abnormal hemoglobins, espe-
cially in the regions in which these types of hemoglobins are 
prevalent.
Hb Beograd cases show two different beta globin gene 
cluster haplotypes, as observed in Turkey. In the first case, Hb 
Beograd mutation is linked with the Mediterranean haplotype I 
[+----++] deduced from the family study. In the second case, 
although the family study could not be applied, the first three 
loci of the 5’-haplotype had a different structure compared to 
the Mediterranean haplotype I. These results show the different 
beta globin gene cluster haplotypes of the Hb Beograd cases 
in Turkey. Since there is no information about the previously 
published cases, the case could not be compared with the 
other Hb Beograd cases regarding their genetic relationships. 
Four different beta globin gene cluster haplotypes for the Hb 
D-Los Angeles mutation have been published [6,8-12]. In this 
study, we report the novel haplotype of [--+-+++]. The beta 
globin gene cluster haplotypes for the Hb D-Los Angeles and 
Hb Beograd in world populations are summarized in Table 2. 
The most common haplotype linked with the Hb D-Los Angeles 
in the world is Mediterranean haplotype I, as in Turkey. Three 
different haplotypes, [+----++], [-++-+++] and [-+--+++], were 
reported from different populations in Italy, Thailand, Mexico, 
Iran and Turkey [6,8-12]. The Asian and Mediterranean con-
nections could be deduced only for the Mediterranean haplo-
type I and Thai haplotype, but the other two haplotypes are 
observed only in Turkey. These uncommon haplotypes could 
reflect different genetic origins and/or other molecular events 
like gene conversions or gene rearrangements. The presence 
of the other reported Turkish haplotypes should be studied in 
more detail at the molecular level in order to understand their 
mutation age, mutation mechanisms and genetic linkages. 
In conclusion, laboratory analysis of the abnormal hemoglo-
bins has a complex structure at the molecular level, emphasiz-
ing the importance of DNA sequencing, especially in the pre-
Table 2. Beta globin gene cluster haplotypes in association with Hb D-Los Angeles and Hb Beograd mutations observed in world populations
 Mutation 5’-ε Gγ Aγ  5’-ψβ 3’-ψβ 5’-β 3’-β Ref.
  Hinc II Hind III Hind III Hinc II Hinc II Ava II Hinf I 
Hb D-Los Angeles        
Italy + - - - - + + [09]
-Thailand - + + - + + + [10]
-Mexico + - - - - + + [11]
-Iran + - - - - + + [12]
-Turkey + - - - - + + [06]
-Turkey - + - - + + + [06]
-Turkey - + + - + + + [08]
-Turkey - - + - + + + This study
 Hb Beograd        
-Turkey + - - - - + + This study
-Turkey - + - +/- +/- + +/- This study
Bahadır et al.
Hb Beograd and Hb D-Los Angeles: Laboratory diagnosis and genetic originsTurk J Hematol 2009; 26: 17-20 19
Figure 1. High performance liquid chromatography profile of Hb Beograd
45.0
37.5
30.0
22.5
15.0
7.5
0.0
1 2
Time (min.)
3 4 5 6
marital screening programs leading to prenatal diagnosis. In 
the case of prenatal diagnosis, molecular identification of the 
abnormal hemoglobins is an important issue in the genetic 
counseling, especially in regions like the Denizli province of 
Turkey. As far as genetic linkages of the beta globin mutations 
and the beta globin gene cluster haplotypes are concerned, 
this should be investigated with more genetic data to deter-
mine their connections and mutation mechanisms.
Acknowledgements 
This study was supported by Pamukkale University 
Research Fund, Project Nos. 2005SBE001 and 2005SBE002.
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Hb Beograd and Hb D-Los Angeles: Laboratory diagnosis and genetic origins Turk J Hematol 2009; 26: 17-2020


Hb D-Los Angeles [beta121(GH4)Glu>Gln] and Hb Beograd [beta121(GH4)Glu>Val]: Implications for their laboratory diagnosis and genetic origins

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Hb D-Los Angeles [beta121(GH4)Glu>Gln] and Hb Beograd [beta121(GH4)Glu>Val]: Implications for their laboratory diagnosis and genetic origins

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