Blood Cells, Molecules, and Diseases 44 (2010) 140145

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Blood Cells, Molecules, and Diseases

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d

Interactions of hemoglobin Lepore ( hybrid hemoglobin) with various

hemoglobinopathies: A molecular and hematological characteristics and
differential diagnosis
Attawut Chaibunruang a,b, Hataichanok Srivorakun a,b, Supan Fucharoen b,, Goonnapa Fucharoen b,
Nattaya Sae-ung b, Kanokwan Sanchaisuriya b
a
Biomedical Sciences Program, Graduate School, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
b
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Hemoglobin (Hb) Lepore is a variant consisting of two -globin and two -globin chains. In heterozygote, it
Submitted 18 November 2009 is associated with clinical ndings of thalassemia minor but interactions with other hemoglobinopathies can
lead to various clinical phenotypes. Using a combination of Hb-HPLC, Hb-capillary electrophoresis and DNA
(Communicated by M. Lichtman, M.D.,
analyses, we have identied 14 patients with Hb LeporeHollandia including eight heterozygotes, two
20 November 2009)
double heterozygotes with +-thalassemia, two compound heterozygotes with Hb E (initially diagnosed as
Keywords:
Hb E--thalassemia) and two previously undescribed conditions of double heterozygote for Hb Lepore/Hb
Hb LeporeHollandia Constant Spring and Hb Lepore/0-thalassemia, both associated with higher levels of Hb F and lower levels
Hb LeporeWashingtonBoston of Hb Lepore. Hematological and molecular features of these patients are presented along with those
Thalassemia observed in four other Thai individuals encountered with heterozygous Hb LeporeWashingtonBoston.
Laboratory diagnosis Haplotype analysis of the -globin gene cluster showed that all Hb LeporeHollandia genes were associated
Beta globin haplotype with a single haplotype not described previously in other populations, ( + + + +) whereas the four
Hb LeporeWashingtonBoston genes were associated with haplotypes (+ + /+) (N = 1) and
(+ +) (N = 3), data indicating multiple origins of these two variants. Hb Lepore may not be
uncommon in the Thai and other Asian populations and both hematological and molecular studies are
required for accurate diagnosis. To facilitate rapid epidemiological, diagnostic screening and differentiation
of the two Hb Lepore defects, a simple assay based on multiplex PCR has been developed.
2009 Elsevier Inc. All rights reserved.

Introduction
Thalassemia and hemoglobinopathies are common in Thailand,
with 2030% of the population having -thalassemia trait, 39% with
-thalassemia trait, and 2030% Hb E trait. The frequency of Hb
Constant Spring or Hb Paks has been found to be at least 4%. Other
hemoglobinopathies have also been reported occasionally. With this
variety of thalassemic alleles, interactions between them results in
complex thalassemia syndromes are common [1,2]. Hb Lepore [2
()2], a structurally abnormal Hb in which the abnormal globin chain
is a hybrid globin chain, can be found in many ethnic groups,
especially in southern European countries. The fusion gene is caused
by unequal crossing over from misaligned - and -globin genes
between two -globin clusters resulting in a deletion of approxi-
mately 7.4 kb, leading to a hybrid gene which produces the hybrid -
Corresponding author. Fax: +66 43 202 083.
E-mail address: supan@kku.ac.th (S. Fucharoen).
globin chain [3]. Three different Lepore Hbs have been dened based
on their deletion breakpoints, namely, Hb LeporeHollandia (22Ala/
50Thr), Hb LeporeBaltimore (50Ser/86Ala or 68Leu/84Thr or
59Lys/86Ala) and Hb LeporeWashingtonBoston (87Gln/IVSII-
8 or 87Gln/116His) [4,5]. The hybrid gene causes a reduced
synthesis of the hybrid chain and produces a -thalassemia
phenotype with mild hypochromic microcytic anemia. Homozygosity
of Hb Lepore or compound heterozygosity of -thalassemia and Hb
Lepore results in thalassemia major or intermedia phenotype. Hb
Lepore has rarely been detected in Thailand and other Southeast Asian
countries. Using a combination of Hb analysis by HPLC and capillary
zone electrophoresis and DNA analysis, we have now described
molecular and hematological features associated with 14 Hb Lepore
Hollandia and 4 Hb LeporeWashingtonBoston observed in Thai
individuals with various genotypes including the previously unde-
scribed conditions. Associated -globin gene haplotypes of these Thai
Hb Lepore genes and a multiplex PCR assay specically developed for
rapid differential diagnosis of these two forms of Hb Lepore are also
presented.

1079-9796/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcmd.2009.11.008
 A. Chaibunruang et al. / Blood Cells, Molecules, and Diseases 44 (2010) 140145 141

Materials and methods
Subjects
We studied 18 unrelated adult Thai patients with initially
unknown abnormal Hbs and elevated Hb F levels, selectively recruited
from our ongoing thalassemia screening program at Khon Kaen
University, Khon Kaen, Thailand. Ethical approval of the study
protocol was obtained from the Institutional Review Board (IRB) of
Khon Kaen University (HE522259). After informed consent was
obtained, peripheral blood samples were collected with EDTA as
anticoagulant. Genomic DNA was prepared from peripheral blood
leukocytes using standard protocols [6].
Hematological and DNA analyses
Initial screening for thalassemia and hemoglobinopathies is
performed routinely using the osmotic fragility (OF) and dichlor-
ophenolindophenol (DCIP) test described elsewhere [7,8]. Hemato-
logical parameters were obtained using a standard automated blood
cell counter (Coulter T series; Beckman-Coulter Co., USA). Hb analysis
was performed using cellulose acetate electrophoresis (CAE) in an
alkaline condition, automated Hb-HPLC analyzer (Variant; Bio-Rad
Laboratories, Hercules, CA, USA) and automated capillary zone
electrophoresis (CapillaryS 2: Sebia, Lisses, France) (Fig. 1) [9].
Identications of the 0-thalassemia (SEA and THAI deletions), +-
thalassemia (3.7 and 4.2 kb deletions), Hb Constant Spring and Hb
Paks were routinely performed in our laboratory using PCR methods
described elsewhere [10-12]. Analysis of -globin gene haplotype
including 7 polymorphic sites; 5 HincII site, G and A HindIII sites,
and 3 HincII sites, AvaII site and 3 BamHI site, was carried
out using PCR and restriction digestion as has been previously
described [13]. The 158 (C-T) G XmnI polymorphism was
examined by PCR amplication of G-globin gene promoter using
primers 4 (5-GGCCTAAAACCACAGAGAGT-3) and 5 (5-CCA-
GAAGCGAGTGTGTGGAA-3) [14] followed by digestion with the

Fig. 1. Representative Hb analysis demonstrating Hb Lepore variant using automated HPLC (A and B) and capillary electrophoresis (C and D). (A and C) Hb Lepore heterozygote,
(B and D) compound Hb Lepore/Hb E. The Hb Lepore with similar retention time with Hb A2/Hb E on HPLC analysis, was clearly observed as the denatured Hb E in zone 6 of the
capillary electrophoresis system.
142 A. Chaibunruang et al. / Blood Cells, Molecules, and Diseases 44 (2010) 140145

Fig. 2. DNA sequencing of the amplied fragments showing the deletion breakpoints of Hb LeporeHollandia (IVS I-42IVS I-56
hybrid) (A) and Hb LeporeWashingtonBoston
(87IVS II-8 hybrid) (B) detected in Thai patients. The crossover region in each Hb Lepore is located within the shaded box.

XmnI restriction enzyme. The crossover breakpoints of the -hybrid
genes were determined on an automated DNA sequencer using ABI
Prism cycle sequencing dye primer ready reaction kit according to the
manufacturer's protocol (Perkin Elmer Biosystems, Norwalk, CT, USA)
(Fig. 2).
Multiplex GAP-PCR for differential diagnosis of Hb Lepore
A scheme for detection of the two forms of Hb Lepore is shown in
Fig. 3. The Hb Leporespecic primers, G58 (5-AGGGCAAGTTAAGG-
GAATAG-3), G59 (5-GCTCACTCAGTGTGGCAAAG-3) and G8 (5-

Fig. 3. Differential diagnosis of Hb Lepore mutations by a multiplex PCR assay. (A) Schematic diagram showing locations and orientations of primers (G58 and G8), (G58 and G59)
and (G7 and G8) that produce three DNA fragments of 1,442 bp specic for hybrid gene, 599 bp Hb LeporeHollandia specic and 213 bp internal control. (B) A representative gel
electrophoresis. Lanes 1 and 6: Normal controls, lanes 2, 4 and 5: Hb LeporeHollandia carriers, lanes 3 and 7: Hb LeporeBoston carriers; M represents the /HindIII size markers.
 A. Chaibunruang et al. / Blood Cells, Molecules, and Diseases 44 (2010) 140145 143

GCTTGGACTCAGAATAATCC-3), and an additional internal control
primer, G7 (5-ATACAATGTATCATGCCTCT-3) were used in a single
multiplex PCR assay. Each PCR reaction (50 l) contained 1 l of DNA,
0.6 pmol of primer G58 and G59, 0.3 pmol of primer G8 and 0.15 pmol
of primer G7, 200 M dNTPs, 10 mM TrisHCl pH 8.3, 50 mM KCl,
0.01% gelatin, 1.5 mM MgCl2, 1 M betaine and 12 units of Taq DNA
polymerase (New England Biolabs Inc., MA, USA). After heating at
94 C for 3 min, PCR was carried out in the GeneAmp PCR 9600 system
(Perkin-Elmer co., Wellesley, MA, USA) as follows: 10 cycles of (94 C
for 1 min, 60 C for 1 min and 72 C for 1 min) and 30 cycles of (94 C
for 1 min, 55 C for 1 min and 72 C for 1 min) with a nal extension
at 72 C for 10 min. The amplied product was separated on 1.5%
agarose gel electrophoresis, stained with ethidium bromide (0.5 g/
ml) and visualized under UV-light. Under this newly developed
PCR system, the size of specic fragment for the two forms of Hb
Lepore is 1442 bp generated by primers (G58 and G8). Further
differential diagnosis can be made by the 599 bp fragment
generated using primers (G58 and G59) which is specic for the
Hb LeporeHollandia. The 213 bp -globin specic fragment
generated from primers (G7 and G8) is an internal control for the
PCR system.
Results
Table 1 lists the laboratory ndings for 18 Thai patients with Hb
Lepore hemoglobinopathies. All presented with mild hypochromic
microcytosis with Hb levels ranging from 9.7 to 12.9 g/dL, MCV 66.6 to
79.0 fL and MCH 19.9 to 26.5 pg. Abnormal Hbs migrating in a similar
position to Hb S on alkaline cellulose acetate electrophoresis in
alkaline pH were observed in all cases. On Hb-HPLC analysis
(Variant; Bio-Rad Laboratories) this abnormal Hb was co-eluted
with Hb A2 and Hb E (Fig. 1; A and B). However, on the capillary zone
electrophoresis system (Capillarys 2; Sebia) which can report Hb A2 in
the presence of Hb E, it was clearly separated as the denatured Hb E
peak in zone 6 (Fig. 1; C and D). The amount of each Hb was therefore
reported using this capillary zone electrophoresis system. The Hb A2
levels were within the normal range and slight elevation of Hb F (3.1
15.7%) were observed for all cases, but there were two compound
heterozygotes with Hb E (initially diagnosed as the Hb E/-
thalassemia disease patients) whose Hb F levels were found to be
46.5 and 41.6%, respectively. In these two cases, in addition to the
abnormal Hbs observed, 46.5% and 49.8% Hb E were detected. The
amounts of abnormal Hbs segregating at the denatured Hb E in zone 6
varied between 4.4% and 13.0%. Further DNA analysis of and -
globin genes by PCR and direct DNA sequencing identied that the
abnormal Hbs in these Thai patients were caused by two different Hb
Lepore mutations i.e. the Hb LeporeHollandia with the IVS I-42/IVS I-56
breakpoint, differing from most reported cases with the 22/
IVS1#16 breakpoint and the Hb LeporeWashingtonBoston with the
87/IVS II-8 breakpoint as shown in Fig. 2, leading to the synthesis of
hybrid Hb molecules. Fourteen patients were found to carry the Hb
LeporeHallandia and the other 4 patients were heterozygotes for Hb
LeporeWashingtonBoston. Routine DNA analysis of common -
thalassemia genes in Thailand further identied that among 14 Hb
LeporeHollandia carriers, two were double heterozygotes for Hb
Lepore/+-thalassemia (3.7 kb deletion), and one patient each carried
a hitherto un-described condition; double heterozygote for Hb
Lepore/Hb Constant Spring and a double heterozygote for Hb
Lepore/0 -thalassemia (SEA deletion). No -thalassemia was
detected among 4 carriers with Hb LeporeWashingtonBoston.
To conrm these ndings and also to provide a rapid DNA assay for
differential diagnosis of these two hemoglobinopathies, we have
developed a multiplex PCR method as shown in Fig. 3. With this
multiplex PCR system, the 213-bp fragment generated with primers
(G7 and G8) is used as an internal control of the PCR system whereas
the 1442 bp fragment produced by primers (G58 and G8) indicates
the presence of the -hybrid Hb Lepore mutation. The 599-bp
fragment generated with primers (G58 and G59) is used to specify
simultaneously the Hb LeporeHollandia type. Using this method of
DNA analysis, all 14 Hb LeporeHollandia carriers and 4 Hb Lepore
WashingtonBoston were correctly conrmed. As Hb Lepore types
have been found in diverse populations, we further established the -
globin gene haplotypes associated with the two Hb Lepore genes in
these Thai patients. With this analysis, it was found that all the Thai
Hb LeporeHollandia chromosomes were associated with an identical
haplotype; ( + + + +) whereas the Hb LeporeWashington
Boston genes were associated with two other haplotypes; 1 with
(+ + /+) and 3 with (+ +), the data

Table 1
Hematological data of 18 Thai patients with Hb Lepore in various combinations. Values are presented as mean standard deviation or as raw data where appropriate. LH; -Lepore
Hollandia, WB; -WashingtonBoston, -SEA; 0 -thalassemia (SEA deletion), -3.7; + -thalassemia (3.7 kb deletion), CS; Hb Constant Spring.

Parameters Heterozygous Hb Heterozygous Hb Heterozygous Hb Heterozygous Hb Heterozygous Hb Heterozygous Hb

LeporeHollandia LeporeHollandia with LeporeHollandia with LeporeHollandia with LeporeHollandia LeporeWashingtonBoston
+-thalassemia Hb constant spring 0-thalassemia with Hb E

No 8 2 1 1 2 4
-genotype A/LH A/LH A/LH A/LH E/LH A/WB
-genotype / 3.7/ CS/ SEA/ / /

Rbc (1012/L) 5.8 0.6 4.4, 4.6 5.1 4.9 4.3, 4.0 5.7 0.6
Hb (g/dL) 12.3 0.7 10.2, 12.1 11.9 10.9 10.3, 9.7 11.3 0.6
Hct (%) 40.2 3.7 32.0, 36.0 35.7 34.9 29.1, 28.0 37.9 2.7
MCV (fL) 70.2 1.7 71.1, 79.0 70.4 70.0 68.1, 69.8 66.6 3.0
MCH (pg) 21.4 0.9 22.9, 26.5 23.5 21.9 24.1, 24.2 19.9 1.6
MCHC (g/dL) 30.8 1.2 33.3, 34.2 33.3 31.3 35.4, 34.6 29.9 2.0
RDW-CV (%) 18.8 0.4 16.5, 15.4 17.0 16.5 22.8, 22.0 17.1 0.6

Hb A2 (%)a 2.3 0.3 2.3, 2.1 2.2 1.8 2.6, 2.2 2.4 0.2
Hb F (%)a 4.0 1.9 5.5, 6.2 15.7 11.8 46.5, 49.8 3.1 1.4
Hb Lepore (%)a 11.7 1.0 10.8, 11.5 8.7 4.4 8.9, 6.4 10.4 0.2
Hb E (%)a None none none none 42.0, 41.6 None

158 bp G XmnI +/+ (6), +/ (2) +/ +/ +/+ +/+ +/ (1), / (3)

b
-Lepore haplotype ( + + + +) ( + + + +) ( + + + +) ( + + + +) ( + + + +) (+ + +/) (1)
(+ +) (3)
a
Determined by the capillary zone electrophoresis (Sebia).
b
Including 7 polymorphic sites: -HincII, G-HindIII, A-HindIII, -HincII, 3-HincII, -AvaII and 3-BamHI.
144 A. Chaibunruang et al. / Blood Cells, Molecules, and Diseases 44 (2010) 140145
presented for the rst time in the Thai population and are useful for
future genetic study of this common hemoglobinopathy.
Discussion
Hb Lepore is a heterogeneous group of thalassemia-like disorders
caused by the fusion of - and -globin genes resulting in the
synthesis of -hybrid globin chain consisting of the N-terminal end
of -globin and C-terminal end of -globin chains. Three known
structural variants of Hb Lepore differing in the position at which the
deletion breakpoint occurs have been described with similar
electrophoretic and chromatographic properties [3]. The crossover
region generating Hb LeporeWashingtonBoston has been localized
to being between 87/IVS2#8 or 87/116. The breakpoints for Hb
LeporeHallandia and Hb Lepore Baltimore have been dened as
being between codon 22/50, and 50/ 86, 68/ 84 or 59/ 86,
respectively [4,5,15]. Recently, a more complex rearrangement of /
globin genes causing another Hb Lepore variant, the Hb Lepore
Leiden has also been reported [16]. The hybrid globin chain is
inefciently synthesized because the promoter of this gene is in
origin which lacks the CCAAT box as well as lacking the CACC box that
binds the -globin specic transcription factor, EKLF [17]. This
reduced synthesis of -chain is the basis for -thalassemia
phenotype associated with this variant. Heterozygotes for Hb Lepore
usually present with mild anemia, hypochromic microcytosis and
increased Hb F [3]. In homozygous or compound heterozygous states
with other thalassemia and hemoglobinopathies including Hb S and
Hb E phenotypes, there can be variation from mild thalassemia
intermedia to thalassemia major [18-23]. Among the three variants,
Hb LeporeWashingtonBoston and Hb LeporeBaltimore are most
common and have a worldwide distribution in many different ethnic
groups including southern European, Asian Indians and African
Americans whereas Hb LeporeHollandia has been thought to be a
rare variant [24-27]. Hb Lepore has rarely been found in Southeast
Asian countries although sporadic cases of Hb Lepore heterozygotes
and compound heterozygote for Hb Lepore/Hb E have been
reported in Thailand and Malaysia [28-30]. Little data on DNA
analysis exist.
Identication of Hb LeporeHollandia and Hb LeporeWashington
Boston in 18 unrelated Thai patients in this study indicates that this
variant is not uncommon in Southeast Asian populations. It is
noteworthy that the crossover region of all Hb LeporeHollandia genes
encountered in this and another study with the (IVS I-42/IVS I-56)
breakpoint [31] differs from that described previously in most reported
cases of Hb LeporeHollandia (22/IVS1#16) [20,22,29], although the
resultant hybrid Hb Lepore molecules are identical. In addition, as
shown in Table 1, different haplotype backgrounds of the Hb Lepore
Hollandia were observed i.e. haplotype ( + + + +) in this study
and (+ nd) in another study [29]. These likely indicate two
independent mutational events of the Hb LeporeHollandia in Southeast
Asian populations. The same nding was observed for the Hb Lepore
WashingtonBoston. We detected two different haplotypes associated
with the Hb LeporeWashingtonBoston among 4 Thai individuals
examined. Identication of Hb LeporeHollandia and Hb Lepore
WashingtonBoston on different haplotypes in these Thai patients
conrms the previous suggestion of a multicentric origin of this hybrid
Hb variant [32,33].
Hematological ndings of these Thai individuals with Hb Lepore
shown in Table 1 conrm that this Hb variant exhibits a hematological
picture quite similar to that of -thalassemia with mild hypochromic
microcytosis. All heterozygotes of both types of Hb Lepore are healthy
and have normal Hb A2 levels. Hb F is slightly elevated. The useful
marker that facilitates the identication of Hb Lepore is the shorter
retention time in HPLC of Hb Lepore, which elutes at 3.343.42 min
whilst that of Hb A2 is 3.593.65 min [26], therefore appearing as a
single peak with small shoulder in the A2 window (Fig. 1A) but this is
not useful in cases with compound Hb Lepore/Hb E (Fig. 1B). On the
contrary, initial diagnosis is relatively simpler with the capillary
electrophoresis system since this Hb variant is clearly separated as a
peak of denatured Hb E in zone 6. However, further DNA conrmatory
testing is still required. We observe that not all cases with this
denatured Hb E peak on the capillary electrophoresis system carry the
Hb Lepore mutation (data not shown).
The association of Hb LeporeHollandia and +-thalassemia
(3.7 kb deletion) has so far been rarely reported [22]. As shown in
Table 1, our 2 patients with this genotype had very similar
hematological phenotype to that of the Hb Lepore heterozygote.
Apparently, the coexistence of +-thalassemia with Hb Lepore does
not contribute further to the severity of the patient's symptom. As
compared to the Hb Lepore heterozygote and a double Hb Lepore/+-
thalassemia (-3.7), we observed higher Hb F levels in the two
previously un-described conditions; Hb Lepore/Hb Constant Spring
(Hb F 15.7%) and Hb Lepore/0-thalassemia (Hb F 11.8%). It is
noteworthy that in this double heterozygote for Hb Lepore/Hb
Constant Spring, we observed no Hb Constant Spring (CS2A2) or
Hb Constant SpringLepore (CS2LH2) on both HPLC chromatogram
and capillary electrophoregram of the patient. Accurate genotype of
the case was obtained after DNA analysis. Interestingly, in addition to
the higher Hb F levels, these two conditions were associated with
lower outputs of Hb Lepore (8.7% and 4.4%, respectively). Based on the
hematological data observed for the Bangladesh patient with Hb E/Hb
Lepore and +-thalassemia, it has been proposed that when the
availability of -globin chain is decreased in -thalassemia, the
formation of Hb E and Hb Lepore are favored over the formation of Hb
F [22]. This is not the case for our two Thai patients. We observed
higher levels of Hb F than Hb Lepore. However, differences might be
related to the variation in Hb F expression of the patients. It has been
reported in Hb LeporeBaltimore carriers with a high Hb F and low Hb
F phenotype that the XmnI (+) at the 158 G and the (AT)x(T)y
repeat region at 540 bp of the -globin gene in trans to the Lepore
chromosome can account for much of the variability in Hb F level [34].
In agreement with this, we detected the XmnI (+/) in the double
heterozygote for Hb Lepore/Hb Constant Spring with 15.7% Hb F and
XmnI (+/+) in the Hb Lepore/0-thalassemia patient with 11.8% Hb
F. In contrast, the XmnI polymorphism was found to be (+/)
(N = 1) and (/) (N = 3) in 4 carriers of Hb LeporeWashington
Boston who had lower Hb F levels (3.1 1.4%) as shown in Table 1.
The conguration of the (AT)x(T)y repeat region at 540 bp of the -
globin gene in trans to these Lepore chromosomes remains to be
elucidated.
The results in Table 1 conrm that the clinical features and
hematological phenotypes of the two patients with compound Hb
Lepore/Hb E mimic Hb E--thalassemia disease commonly encoun-
tered in northeast Thailand but with relatively milder phenotype [35].
In fact, the two patients were initially diagnosed as having Hb E--
thalassemia disease. Although having hypochromic microcytic ane-
mia, both of them were healthy and had no history of blood
transfusion. Again Hb analysis using the capillary electrophoresis is
helpful in initial recognition of these cases (Fig. 1D), accurate diagnosis
could only be obtained after DNA analysis. This conrms the clinical
phenotype of thalassemia intermedia of the Hb Lepore/Hb E syndrome
which has also been noted in other populations [20,22,23].
It is conceivable that the Hb Lepore variant may not be uncommon
in Thai and other Southeast Asian populations and many cases of Hb
Lepore may remain undetected unless detailed DNA analysis is
performed. Interaction of this abnormal gene with other hemoglo-
binopathies could lead to complex thalassemia syndromes with
varying phenotypic expressions. As shown in Fig. 1, a possible
misdiagnosis of Hb Lepore in heterozygote or compound heterozygote
states with other hemoglobinopathies could occur in a routine setting.
Hb Lepore, Hb A2 and Hb E are not distinctly separated on standard
cation exchange HPLC format, although analysis using the capillary
 A. Chaibunruang et al. / Blood Cells, Molecules, and Diseases 44 (2010) 140145
electrophoresis system is helpful in the initial recognition of Hb
Lepore. Accurate diagnosis of this clinically relevant hemoglobinop-
athy using both hematological and DNA analyses as being performed
in this study would provide information necessary for proper clinical
management, genetic counseling and prenatal diagnosis.
Acknowledgments
The study was supported by grants to S.F. from Khon Kaen University
and Ofce of the Higher Education Commission (CHE-RG-51), Ministry
of Education, Thailand. A.C. and H.S. are supported by the Royal Golden
Jubilee Ph.D. program (PHD/0174/2548 and PHD/0132/2549) of the
Thailand Research Fund (TRF), Thailand. We thank Ian Thomas for
helpful comments on the manuscript.
References
[1] S. Fucharoen, P. Winichagoon, Hemoglobinopathies in Southeast Asia,
Hemoglobin 11 (1987) 6588.
[2] S. Fucharoen, P. Winichagoon, N. Sirithanaratkul, J. Chowthaworn, P. Pootrakul, -
and - thalassemia in Thailand, Ann. N.Y. Acad. Sci 850 (1998) 412414.
[3] D.J. Weatherall, J. Clegg, The Thalassemia Syndromes, 4th edBlackwell Science
Ltd., UK., Oxford, 2001.
[4] A.B. Metzenberg, G. Wurzer, T.H.J Huisman, O. Smithies, Homology requirements
for unequal crossing over in humans, Genetics 128 (1991) 143161.
[5] K.D. Lanclos, J. Patterson, G.D. Efremov, et al., Characterization of chromosomes
with hybrid genes for Hb LeporeWashington, Hb LeporeBaltimore, Hb P-Nilotic,
Hb Kenya, Hum. Genet. 77 (1987) 4045.
[6] S. Fucharoen, G. Fucharoen, W. Sriroongrueng, et al., Molecular basis of -
thalassemia in Thailand: analysis of -thalassemia mutations using the polymer-
ase chain reaction, Hum. Genet. 84 (1989) 4146.
[7] G. Fucharoen, K. Sanchaisuriya, N. Sae-ung, S. Dangwibul, S. Fucharoen, A simplied
screening strategy for thalassaemia and haemoglobin E in rural communities of
Southeast Asia, Bull. World Health Organ. 82 (2004) 364372.
[8] K. Sanchaisuriya, S. Fucharoen, G. Fucharoen, et al., A reliable screening protocol
for thalassemia and hemoglobinopathies in pregnancy; an alternative approach to
electronic blood cell counting, Am. J. Clin. Pathol. 123 (2005) 113118.
[9] H. Srivorakun, G. Fucharoen, N. Sae-ung, K. Sanchaisuriya, T. Ratanasiri, S. Fucharoen,
Analysis of fetal blood using capillary electrophoresis system: a simple method for
prenatal diagnosis of severe thalassemia diseases, Eur. J. Haematol. 83 (2009) 5765.
[10] K. Sanchaisuriya, G. Fucharoen, N. Sae-ung, A. Jetsrisuparb, S. Fucharoen,
Molecular and hematologic features of hemoglobin E heterozygotes with different
forms of -thalassemia in Thailand, Ann. Hematol. 82 (2003) 612616.
[11] S. Boonsa, K. Sanchaisuriya, G. Fucharoen, S. Wiangnon, A. Jetsrisuparb, S.
Fucharoen, The diverse molecular basis and hematologic features of Hb H and
AEBart's diseases in northeast Thailand, Acta Haematol. 111 (2004) 149154.
[12] N. Sae-ung, G. Fucharoen, K. Sanchaisuriya, S. Fucharoen, 0-thalassemia and
related disorders in northeast Thailand: a molecular and hematological
characterization, Acta Haematol. 117 (2007) 7882.
[13] Y. Fukumaki, S. Fucharoen, Generation and spread of globin gene mutations in
populations: -thalassemia in Asian countries, in: M Kimaru, N Takahata (Eds.),
New aspects of the genetics of molecular evolution, Springer-Verlag, Berlin, 1991,
pp. 153176.
145
[14] S. Fucharoen, K. Shimizu, Y. Fukumaki, A novel CT transition within the distal
CCAAT motif of the G-globin gene in the Japanese HPFH: implication of factor
binding in elevated fetal globin expression, Nucleic Acids Res. 18 (1990)
52455253.
[15] T.H.J. Huisman, M.F.H. Carver, E. Baysal, A syllabus of thalassemia mutations, The
Sickle Cell Anemia Foundation, Augusta, GA., 1997.
[16] C.L. Harteveld, P.W. Wijermans, S.G.J. Arkesteijn, P. Van Delft, J.L. Kerkhoffs, P.C.
Giordano, Hb LeporeLeiden: a new / rearrangement associated with a -
thalassemia minor phenotype, Hemoglobin 32 (2008) 446453.
[17] J. Slone-Stanley, N.A. Roberts, N. Olivieri, D.J. Weatherall, W.G. Wood, Globin gene
expression in Hb Lepore-BAC transgenic mice, Br. J. Haematol. 135 (2006)
735737.
[18] E. Voskaridou, K. Konstantopoulos, P. Kollia, M. Papadakis, D. Loukopoulos, Hb
Lepore (Pylos)/Hb S compound heterozygosity in two Greek families, Am. J.
Hematol. 49 (1995) 131134.
[19] D.P. Seward, D.E. Ware, T.R. Kinney, Hemoglobin sickle-Lepore: report of two
siblings and review of the literature, Am. J. Hematol. 44 (1993) 192195.
[20] E.S. Edison, R.V. Shaji, A. Srivastava, M. Chandy, Compound heterozygosity for Hb
E and Hb LeporeHollandia in India; rst report and potential diagnostic pitfalls,
Hemoglobin 29 (2005) 221224.
[21] D.G. Efremov, G.D. Efremov, N. Zisovski, et al., Variation in clinical severity among
patients with Hb LeporeBoston--thalassaemia is related to the type of -
thalassaemia, Br. J. Haematol. 68 (1998) 351355.
[22] J.S. Waye, B. Eng, M. Patterson, et al., Hb E/Hb Lepore Hollandia in a family from
Bangladesh, Am. J. Hematol. 47 (1994) 262265.
[23] V. Sherma, V.P. Choudhry, R. Saxana, Association of Hb E with Hb Lepore and a
triplication in a Bengali family, Clin. Chim. Acta 395 (2008) 175177.
[24] R.V. Shaji, E.S. Edison, R. Krishnamoorthy, M. Chandy, A. Srivastava, Hb Lepore in
the Indian population, Hemoglobin 27 (2003) 714.
[25] M.L. Ribeiro, E. Cunha, P. Goncalves, et al., Hb LeporeBaltimore (68Leu-84Thr)
and Hb LeporeWashingtonBoston (87Gln-IVSII#8) in Central Portugal and
the Spanish Alta Extramadura, Hum. Genet. 99 (1997) 669673.
[26] P. Ropero, F.A. Gonzales, J. Sanchez, et al., Identication of the Hb Lepore
phenotype by HPLC, Haematologica 84 (1999) 10811084.
[27] S.M. McKeown, H. Carmichael, R.B. Markowitz, A. Kutlar, L. Holley, F. Kutlar, Rare
occurrence of Hb LeporeBaltimore in African Americans: molecular character-
istics and variations of Hb Lepore, Ann. Hematol. 88 (2009) 545548.
[28] P. Boontrakoonpoontawee, J. Svasti, S. Fucharoen, P. Winichagoon, Identication
of Hb LeporeWashingtonBoston in association with Hb E [26 (B8) Glu-Lys] in a
Thai female, Hemoglobin 11 (1987) 309316.
[29] V. Viprakasit, P. Pung-Amritt, L. Suwanthon, K. Clark, V.S. Tanphaichitr, Complex
interactions of hybrid haemoglobin (Hb LeporeHollandia), Hb E (26 GA) and
+-thalassaemia in a Thai family, Eur. J. Haematol. 68 (2002) 107111.
[30] J. Pasangna, E. George, M. Nagaratnam, Haemoglobin Lepore in a Malay family: a
case report, Malays. J. Pathol. 27 (2005) 3337.
[31] V. Chan, P. Au, B. Yip, T.K. Chan, A new cross-over region for hemoglobin-Lepore
Hollandia, Haematologica 89 (2004) 610611.
[32] G. Fioretti, M. De Angioletti, F. Masciangelo, et al., Origin heterogeneity of Hb
LeporeBoston gene in Italy, Am. J. Hum. Genet. 50 (1992) 781786.
[33] S. Pavlovic, J. Urosevic, J. Poznanic, et al., Molecular basis of thalassemia
syndromes in Serbia and Montenegro, Acta Haematol. 113 (2005) 175180.
[34] I. Gonaves, A. Henriques, A. Raimundo, et al., Fetal hemoglobin elevation in Hb
Lepore heterozygotes anf its correlation with globin cluster linked determi-
nants, Am. J. Hematol. 69 (2002) 95102.
[35] L. Nuntakarn, S. Fucharoen, G. Fucharoen, K. Sanchaisuriya, A. Jetsrisuparb, S.
Wiangnon, Molecular, hematological and clinical aspects of thalassemia major
and thalassemia intermedia associated with Hb E--thalassemia in northeast
Thailand, Blood Cells Mol. Dis. 42 (2009) 3235.