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Interactions of hemoglobin Lepore (δβ hybrid hemoglobin) with various hemoglobinopathies: A molecular and hematological characteristics and differential diagnosis
Blood Cells, Molecules, and Diseases 44 (2010) 140–145
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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 (δβ 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 b
Biomedical Sciences Program, Graduate School, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand 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
Article history: Submitted 18 November 2009 (Communicated by M. Lichtman, M.D., 20 November 2009) Keywords: Hb Lepore–Hollandia Hb Lepore–Washington–Boston Thalassemia Laboratory diagnosis Beta globin haplotype
a b s t r a c t Hemoglobin (Hb) Lepore is a variant consisting of two α-globin and two δβ-globin chains. In heterozygote, it is associated with clinical findings 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 analyses, we have identified 14 patients with Hb Lepore–Hollandia including eight heterozygotes, two double heterozygotes with α+-thalassemia, two compound heterozygotes with Hb E (initially diagnosed as Hb E-β-thalassemia) and two previously undescribed conditions of double heterozygote for Hb Lepore/Hb Constant Spring and Hb Lepore/α0-thalassemia, both associated with higher levels of Hb F and lower levels of Hb Lepore. Hematological and molecular features of these patients are presented along with those observed in four other Thai individuals encountered with heterozygous Hb Lepore–Washington–Boston. Haplotype analysis of the β-globin gene cluster showed that all Hb Lepore–Hollandia genes were associated with a single haplotype not described previously in other populations, (− + − + + − +) whereas the four Hb Lepore–Washington–Boston 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 20–30% of the population having α-thalassemia trait, 3–9% with β-thalassemia trait, and 20–30% Hb E trait. The frequency of Hb Constant Spring or Hb Paksé 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 [α2 (δβ)2], a structurally abnormal Hb in which the abnormal globin chain is a hybrid δβ 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 δ- and β-globin genes between two β-globin clusters resulting in a deletion of approximately 7.4 kb, leading to a hybrid gene which produces the hybrid δβ-
⁎ Corresponding author. Fax: +66 43 202 083. E-mail address: [email protected] (S. Fucharoen). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2009.11.008
globin chain [3]. Three different Lepore Hbs have been defined based on their deletion breakpoints, namely, Hb Lepore–Hollandia (δ22Ala/ β50Thr), Hb Lepore–Baltimore (δ50Ser/β86Ala or δ68Leu/β84Thr or δ59Lys/β86Ala) and Hb Lepore–Washington–Boston (δ87Gln/βIVSII8 or δ87Gln/β116His) [4,5]. The hybrid gene causes a reduced synthesis of the δβ hybrid chain and produces a β-thalassemia phenotype with mild hypochromic microcytic anemia. Homozygosity of Hb Lepore or compound heterozygosity of β-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 Lepore–Washington–Boston observed in Thai individuals with various genotypes including the previously undescribed conditions. Associated β-globin gene haplotypes of these Thai Hb Lepore genes and a multiplex PCR assay specifically developed for rapid differential diagnosis of these two forms of Hb Lepore are also presented.
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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-
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ophenolindophenol (DCIP) test described elsewhere [7,8]. Hematological 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]. Identifications of the α0-thalassemia (SEA and THAI deletions), α+thalassemia (3.7 and 4.2 kb deletions), Hb Constant Spring and Hb Paksé were routinely performed in our laboratory using PCR methods described elsewhere [10-12]. Analysis of β-globin gene haplotype including 7 polymorphic sites; 5′ɛ HincII site, Gγ and Aγ HindIII sites, ψβ and 3′ψβ HincII sites, β AvaII site and 3′β BamHI site, was carried out using PCR and restriction digestion as has been previously described [13]. The − 158 (C-T) Gγ XmnI polymorphism was examined by PCR amplification of Gγ-globin gene promoter using primers γ4 (5′-GGCCTAAAACCACAGAGAGT-3′) and γ5 (5′-CCAGAAGCGAGTGTGTGGAA-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.
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Fig. 2. DNA sequencing of the amplified fragments showing the deletion breakpoints of Hb Lepore–Hollandia (δIVS I-42βIVS (δ87βIVS 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 δβ-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).
I-56
hybrid) (A) and Hb Lepore–Washington–Boston
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 Lepore–specific primers, G58 (5′-AGGGCAAGTTAAGGGAATAG-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 specific for δβ hybrid gene, 599 bp Hb Lepore–Hollandia specific and 213 bp internal control. (B) A representative gel electrophoresis. Lanes 1 and 6: Normal controls, lanes 2, 4 and 5: Hb Lepore–Hollandia carriers, lanes 3 and 7: Hb Lepore–Boston carriers; M represents the λ/HindIII size markers.
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GCTTGGACTCAGAATAATCC-3′), and an additional internal control primer, G7 (5′-ATACAATGTATCATGCCTCT-3′) were used in a single multiplex PCR assay. Each PCR reaction (50 μl) contained 1 μl of DNA, 0.6 pmol of primer G58 and G59, 0.3 pmol of primer G8 and 0.15 pmol of primer G7, 200 μM dNTPs, 10 mM Tris–HCl pH 8.3, 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl2, 1 M betaine and 1–2 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 final extension at 72 °C for 10 min. The amplified product was separated on 1.5% agarose gel electrophoresis, stained with ethidium bromide (0.5 μg/ ml) and visualized under UV-light. Under this newly developed PCR system, the size of specific 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 specific for the Hb Lepore–Hollandia. The 213 bp β-globin specific fragment generated from primers (G7 and G8) is an internal control for the PCR system. Results Table 1 lists the laboratory findings 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/β-
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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 δ and βglobin genes by PCR and direct DNA sequencing identified that the abnormal Hbs in these Thai patients were caused by two different Hb Lepore mutations i.e. the Hb Lepore–Hollandia with the δIVS I-42/βIVS I-56 breakpoint, differing from most reported cases with the δ22/ βIVS1#16 breakpoint and the Hb Lepore–Washington–Boston with the δ87/βIVS II-8 breakpoint as shown in Fig. 2, leading to the synthesis of δβ hybrid Hb molecules. Fourteen patients were found to carry the Hb Lepore–Hallandia and the other 4 patients were heterozygotes for Hb Lepore–Washington–Boston. Routine DNA analysis of common αthalassemia genes in Thailand further identified that among 14 Hb Lepore–Hollandia carriers, two were double heterozygotes for Hb Lepore/α+-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/α0 -thalassemia (SEA deletion). No α-thalassemia was detected among 4 carriers with Hb Lepore–Washington–Boston. To confirm these findings 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 δβ-hybrid Hb Lepore mutation. The 599-bp fragment generated with primers (G58 and G59) is used to specify simultaneously the Hb Lepore–Hollandia type. Using this method of DNA analysis, all 14 Hb Lepore–Hollandia carriers and 4 Hb Lepore– Washington–Boston were correctly confirmed. As Hb Lepore types have been found in diverse populations, we further established the βglobin gene haplotypes associated with the two Hb Lepore genes in these Thai patients. With this analysis, it was found that all the Thai Hb Lepore–Hollandia chromosomes were associated with an identical haplotype; (− + − + + − +) whereas the Hb Lepore–Washington– 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. βLH; β-Lepore– Hollandia, βWB; β-Washington–Boston, -SEA; α0 -thalassemia (SEA deletion), -α3.7; α+ -thalassemia (3.7 kb deletion), αCS; Hb Constant Spring. Parameters
Heterozygous Hb Lepore–Hollandia
Heterozygous Hb Lepore–Hollandia with α+-thalassemia
Heterozygous Hb Lepore–Hollandia with Hb constant spring
Heterozygous Hb Lepore–Hollandia with α0-thalassemia
Heterozygous Hb Lepore–Hollandia with Hb E
Heterozygous Hb Lepore–Washington–Boston
No β-genotype α-genotype
8 βA/βLH αα /αα
2 βA/βLH − α3.7/ αα
1 βA/βLH αCSα/ αα
1 βA/βLH −−SEA/ αα
2 βE/βLH αα / αα
4 βA/βWB αα / αα
Rbc (1012/L) Hb (g/dL) Hct (%) MCV (fL) MCH (pg) MCHC (g/dL) RDW-CV (%)
5.8 ± 0.6 12.3 ± 0.7 40.2 ± 3.7 70.2 ± 1.7 21.4 ± 0.9 30.8 ± 1.2 18.8 ± 0.4
4.4, 4.6 10.2, 12.1 32.0, 36.0 71.1, 79.0 22.9, 26.5 33.3, 34.2 16.5, 15.4
5.1 11.9 35.7 70.4 23.5 33.3 17.0
4.9 10.9 34.9 70.0 21.9 31.3 16.5
4.3, 4.0 10.3, 9.7 29.1, 28.0 68.1, 69.8 24.1, 24.2 35.4, 34.6 22.8, 22.0
5.7 ± 0.6 11.3 ± 0.6 37.9 ± 2.7 66.6 ± 3.0 19.9 ± 1.6 29.9 ± 2.0 17.1 ± 0.6
2.3 ± 0.3 4.0 ± 1.9 11.7 ± 1.0 None
2.3, 2.1 5.5, 6.2 10.8, 11.5 none
2.2 15.7 8.7 none
1.8 11.8 4.4 none
2.6, 2.2 46.5, 49.8 8.9, 6.4 42.0, 41.6
2.4 ± 0.2 3.1 ± 1.4 10.4 ± 0.2 None
+/+ (6), +/− (2)
+/−
+/−
+/+
+/+
+/− (1), −/− (3)
(− + − + + − +)
(− + − + + − +)
(− + − + + − +)
(− + − + + − +)
(− + − + + − +)
(+ − − − − + +/−) (1) (+ − − − − − +) (3)
Hb Hb Hb Hb
A2 (%)a F (%)a Lepore (%)a E (%)a
− 158 bp Gγ XmnI β-Lepore haplotype a b
b
Determined by the capillary zone electrophoresis (Sebia). Including 7 polymorphic sites: ɛ-HincII, Gγ-HindIII, Aγ-HindIII, ψβ-HincII, 3′Ψβ-HincII, β-AvaII and 3′β-BamHI.
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presented for the first 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 δ- and β-globin genes resulting in the synthesis of δβ-hybrid globin chain consisting of the N-terminal end of δ-globin and C-terminal end of β-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 Lepore–Washington–Boston has been localized to being between δ87/βIVS2#8 or δ87/β116. The breakpoints for Hb Lepore–Hallandia and Hb Lepore Baltimore have been defined as being between codon δ22/β50, and δ50/ β86, δ68/ β84 or δ59/ β86, respectively [4,5,15]. Recently, a more complex rearrangement of δ/β globin genes causing another Hb Lepore variant, the Hb Lepore– Leiden has also been reported [16]. The δβ hybrid globin chain is inefficiently synthesized because the promoter of this gene is δ in origin which lacks the CCAAT box as well as lacking the CACC box that binds the β-globin specific transcription factor, EKLF [17]. This reduced synthesis of δβ-chain is the basis for β-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 Lepore–Washington–Boston and Hb Lepore–Baltimore are most common and have a worldwide distribution in many different ethnic groups including southern European, Asian Indians and African Americans whereas Hb Lepore–Hollandia 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. Identification of Hb Lepore–Hollandia and Hb Lepore–Washington– 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 Lepore–Hollandia genes encountered in this and another study with the (δIVS I-42/βIVS I-56) breakpoint [31] differs from that described previously in most reported cases of Hb Lepore–Hollandia (δ22/βIVS1#16) [20,22,29], although the resultant δβ 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 Lepore–Hollandia in Southeast Asian populations. The same finding was observed for the Hb Lepore– Washington–Boston. We detected two different haplotypes associated with the Hb Lepore–Washington–Boston among 4 Thai individuals examined. Identification of Hb Lepore–Hollandia and Hb Lepore– Washington–Boston on different haplotypes in these Thai patients confirms the previous suggestion of a multicentric origin of this hybrid Hb variant [32,33]. Hematological findings of these Thai individuals with Hb Lepore shown in Table 1 confirm that this Hb variant exhibits a hematological picture quite similar to that of β-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 identification of Hb Lepore is the shorter retention time in HPLC of Hb Lepore, which elutes at 3.34–3.42 min whilst that of Hb A2 is 3.59–3.65 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 confirmatory 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 Lepore–Hollandia and α+-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 α+-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/α+thalassemia (-α3.7), 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/α0-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 (αCS2βA2) or Hb Constant Spring–Lepore (αCS2δβLH2) 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 α+-thalassemia, it has been proposed that when the availability of α-globin chain is decreased in α-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 Lepore–Baltimore carriers with a high Hb F and low Hb F phenotype that the XmnI (+) at the − 158 Gγ and the (AT)x(T)y repeat region at −540 bp of the β-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/α0-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 Lepore–Washington– Boston who had lower Hb F levels (3.1 ± 1.4%) as shown in Table 1. The configuration of the (AT)x(T)y repeat region at −540 bp of the βglobin gene in trans to these Lepore chromosomes remains to be elucidated. The results in Table 1 confirm that the clinical features and hematological phenotypes of the two patients with compound Hb Lepore/Hb E mimic Hb E-β-thalassemia disease commonly encountered in northeast Thailand but with relatively milder phenotype [35]. In fact, the two patients were initially diagnosed as having Hb E-βthalassemia disease. Although having hypochromic microcytic anemia, 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 confirms 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 hemoglobinopathies 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
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electrophoresis system is helpful in the initial recognition of Hb Lepore. Accurate diagnosis of this clinically relevant hemoglobinopathy 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 Office 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) 65–88. [2] S. Fucharoen, P. Winichagoon, N. Sirithanaratkul, J. Chowthaworn, P. Pootrakul, αand β- thalassemia in Thailand, Ann. N.Y. Acad. Sci 850 (1998) 412–414. [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) 143–161. [5] K.D. Lanclos, J. Patterson, G.D. Efremov, et al., Characterization of chromosomes with hybrid genes for Hb Lepore–Washington, Hb Lepore–Baltimore, Hb P-Nilotic, Hb Kenya, Hum. Genet. 77 (1987) 40–45. [6] S. Fucharoen, G. Fucharoen, W. Sriroongrueng, et al., Molecular basis of βthalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction, Hum. Genet. 84 (1989) 41–46. [7] G. Fucharoen, K. Sanchaisuriya, N. Sae-ung, S. Dangwibul, S. Fucharoen, A simplified screening strategy for thalassaemia and haemoglobin E in rural communities of Southeast Asia, Bull. World Health Organ. 82 (2004) 364–372. [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) 113–118. [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) 57–65. [10] K. Sanchaisuriya, G. Fucharoen, N. Sae-ung, A. Jetsrisuparb, S. Fucharoen, Molecular and hematologic features of hemoglobin E heterozygotes with different forms of α-thalassemia in Thailand, Ann. Hematol. 82 (2003) 612–616. [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) 149–154. [12] N. Sae-ung, G. Fucharoen, K. Sanchaisuriya, S. Fucharoen, α0-thalassemia and related disorders in northeast Thailand: a molecular and hematological characterization, Acta Haematol. 117 (2007) 78–82. [13] Y. Fukumaki, S. Fucharoen, Generation and spread of globin gene mutations in populations: β-thalassemia in Asian countries, in: M Kimaru, N Takahata (Eds.), New aspects of the genetics of molecular evolution, Springer-Verlag, Berlin, 1991, pp. 153–176.
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[14] S. Fucharoen, K. Shimizu, Y. Fukumaki, A novel C–T transition within the distal CCAAT motif of the Gγ-globin gene in the Japanese HPFH: implication of factor binding in elevated fetal globin expression, Nucleic Acids Res. 18 (1990) 5245–5253. [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 Lepore–Leiden: a new δ/β rearrangement associated with a βthalassemia minor phenotype, Hemoglobin 32 (2008) 446–453. [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) 735–737. [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) 131–134. [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) 192–195. [20] E.S. Edison, R.V. Shaji, A. Srivastava, M. Chandy, Compound heterozygosity for Hb E and Hb Lepore–Hollandia in India; first report and potential diagnostic pitfalls, Hemoglobin 29 (2005) 221–224. [21] D.G. Efremov, G.D. Efremov, N. Zisovski, et al., Variation in clinical severity among patients with Hb Lepore–Boston-β-thalassaemia is related to the type of βthalassaemia, Br. J. Haematol. 68 (1998) 351–355. [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) 262–265. [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) 175–177. [24] R.V. Shaji, E.S. Edison, R. Krishnamoorthy, M. Chandy, A. Srivastava, Hb Lepore in the Indian population, Hemoglobin 27 (2003) 7–14. [25] M.L. Ribeiro, E. Cunha, P. Goncalves, et al., Hb Lepore–Baltimore (δ68Leu-β84Thr) and Hb Lepore–Washington–Boston (δ87Gln-βIVSII#8) in Central Portugal and the Spanish Alta Extramadura, Hum. Genet. 99 (1997) 669–673. [26] P. Ropero, F.A. Gonzales, J. Sanchez, et al., Identification of the Hb Lepore phenotype by HPLC, Haematologica 84 (1999) 1081–1084. [27] S.M. McKeown, H. Carmichael, R.B. Markowitz, A. Kutlar, L. Holley, F. Kutlar, Rare occurrence of Hb Lepore–Baltimore in African Americans: molecular characteristics and variations of Hb Lepore, Ann. Hematol. 88 (2009) 545–548. [28] P. Boontrakoonpoontawee, J. Svasti, S. Fucharoen, P. Winichagoon, Identification of Hb Lepore–Washington–Boston in association with Hb E [β26 (B8) Glu-Lys] in a Thai female, Hemoglobin 11 (1987) 309–316. [29] V. Viprakasit, P. Pung-Amritt, L. Suwanthon, K. Clark, V.S. Tanphaichitr, Complex interactions of δβ hybrid haemoglobin (Hb Lepore–Hollandia), Hb E (β26 GàA) and α+-thalassaemia in a Thai family, Eur. J. Haematol. 68 (2002) 107–111. [30] J. Pasangna, E. George, M. Nagaratnam, Haemoglobin Lepore in a Malay family: a case report, Malays. J. Pathol. 27 (2005) 33–37. [31] V. Chan, P. Au, B. Yip, T.K. Chan, A new cross-over region for hemoglobin-Lepore– Hollandia, Haematologica 89 (2004) 610–611. [32] G. Fioretti, M. De Angioletti, F. Masciangelo, et al., Origin heterogeneity of Hb Lepore–Boston gene in Italy, Am. J. Hum. Genet. 50 (1992) 781–786. [33] S. Pavlovic, J. Urosevic, J. Poznanic, et al., Molecular basis of thalassemia syndromes in Serbia and Montenegro, Acta Haematol. 113 (2005) 175–180. [34] I. Gonçaves, A. Henriques, A. Raimundo, et al., Fetal hemoglobin elevation in Hb Lepore heterozygotes anf its correlation with β globin cluster linked determinants, Am. J. Hematol. 69 (2002) 95–102. [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-β-thalassemia in northeast Thailand, Blood Cells Mol. Dis. 42 (2009) 32–35.
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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 (δβ 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 b
Biomedical Sciences Program, Graduate School, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand 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
Article history: Submitted 18 November 2009 (Communicated by M. Lichtman, M.D., 20 November 2009) Keywords: Hb Lepore–Hollandia Hb Lepore–Washington–Boston Thalassemia Laboratory diagnosis Beta globin haplotype
a b s t r a c t Hemoglobin (Hb) Lepore is a variant consisting of two α-globin and two δβ-globin chains. In heterozygote, it is associated with clinical findings 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 analyses, we have identified 14 patients with Hb Lepore–Hollandia including eight heterozygotes, two double heterozygotes with α+-thalassemia, two compound heterozygotes with Hb E (initially diagnosed as Hb E-β-thalassemia) and two previously undescribed conditions of double heterozygote for Hb Lepore/Hb Constant Spring and Hb Lepore/α0-thalassemia, both associated with higher levels of Hb F and lower levels of Hb Lepore. Hematological and molecular features of these patients are presented along with those observed in four other Thai individuals encountered with heterozygous Hb Lepore–Washington–Boston. Haplotype analysis of the β-globin gene cluster showed that all Hb Lepore–Hollandia genes were associated with a single haplotype not described previously in other populations, (− + − + + − +) whereas the four Hb Lepore–Washington–Boston 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 20–30% of the population having α-thalassemia trait, 3–9% with β-thalassemia trait, and 20–30% Hb E trait. The frequency of Hb Constant Spring or Hb Paksé 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 [α2 (δβ)2], a structurally abnormal Hb in which the abnormal globin chain is a hybrid δβ 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 δ- and β-globin genes between two β-globin clusters resulting in a deletion of approximately 7.4 kb, leading to a hybrid gene which produces the hybrid δβ-
⁎ Corresponding author. Fax: +66 43 202 083. E-mail address: [email protected] (S. Fucharoen). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2009.11.008
globin chain [3]. Three different Lepore Hbs have been defined based on their deletion breakpoints, namely, Hb Lepore–Hollandia (δ22Ala/ β50Thr), Hb Lepore–Baltimore (δ50Ser/β86Ala or δ68Leu/β84Thr or δ59Lys/β86Ala) and Hb Lepore–Washington–Boston (δ87Gln/βIVSII8 or δ87Gln/β116His) [4,5]. The hybrid gene causes a reduced synthesis of the δβ hybrid chain and produces a β-thalassemia phenotype with mild hypochromic microcytic anemia. Homozygosity of Hb Lepore or compound heterozygosity of β-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 Lepore–Washington–Boston observed in Thai individuals with various genotypes including the previously undescribed conditions. Associated β-globin gene haplotypes of these Thai Hb Lepore genes and a multiplex PCR assay specifically developed for rapid differential diagnosis of these two forms of Hb Lepore are also presented.
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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-
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ophenolindophenol (DCIP) test described elsewhere [7,8]. Hematological 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]. Identifications of the α0-thalassemia (SEA and THAI deletions), α+thalassemia (3.7 and 4.2 kb deletions), Hb Constant Spring and Hb Paksé were routinely performed in our laboratory using PCR methods described elsewhere [10-12]. Analysis of β-globin gene haplotype including 7 polymorphic sites; 5′ɛ HincII site, Gγ and Aγ HindIII sites, ψβ and 3′ψβ HincII sites, β AvaII site and 3′β BamHI site, was carried out using PCR and restriction digestion as has been previously described [13]. The − 158 (C-T) Gγ XmnI polymorphism was examined by PCR amplification of Gγ-globin gene promoter using primers γ4 (5′-GGCCTAAAACCACAGAGAGT-3′) and γ5 (5′-CCAGAAGCGAGTGTGTGGAA-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.
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Fig. 2. DNA sequencing of the amplified fragments showing the deletion breakpoints of Hb Lepore–Hollandia (δIVS I-42βIVS (δ87βIVS 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 δβ-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).
I-56
hybrid) (A) and Hb Lepore–Washington–Boston
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 Lepore–specific primers, G58 (5′-AGGGCAAGTTAAGGGAATAG-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 specific for δβ hybrid gene, 599 bp Hb Lepore–Hollandia specific and 213 bp internal control. (B) A representative gel electrophoresis. Lanes 1 and 6: Normal controls, lanes 2, 4 and 5: Hb Lepore–Hollandia carriers, lanes 3 and 7: Hb Lepore–Boston carriers; M represents the λ/HindIII size markers.
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GCTTGGACTCAGAATAATCC-3′), and an additional internal control primer, G7 (5′-ATACAATGTATCATGCCTCT-3′) were used in a single multiplex PCR assay. Each PCR reaction (50 μl) contained 1 μl of DNA, 0.6 pmol of primer G58 and G59, 0.3 pmol of primer G8 and 0.15 pmol of primer G7, 200 μM dNTPs, 10 mM Tris–HCl pH 8.3, 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl2, 1 M betaine and 1–2 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 final extension at 72 °C for 10 min. The amplified product was separated on 1.5% agarose gel electrophoresis, stained with ethidium bromide (0.5 μg/ ml) and visualized under UV-light. Under this newly developed PCR system, the size of specific 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 specific for the Hb Lepore–Hollandia. The 213 bp β-globin specific fragment generated from primers (G7 and G8) is an internal control for the PCR system. Results Table 1 lists the laboratory findings 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/β-
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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 δ and βglobin genes by PCR and direct DNA sequencing identified that the abnormal Hbs in these Thai patients were caused by two different Hb Lepore mutations i.e. the Hb Lepore–Hollandia with the δIVS I-42/βIVS I-56 breakpoint, differing from most reported cases with the δ22/ βIVS1#16 breakpoint and the Hb Lepore–Washington–Boston with the δ87/βIVS II-8 breakpoint as shown in Fig. 2, leading to the synthesis of δβ hybrid Hb molecules. Fourteen patients were found to carry the Hb Lepore–Hallandia and the other 4 patients were heterozygotes for Hb Lepore–Washington–Boston. Routine DNA analysis of common αthalassemia genes in Thailand further identified that among 14 Hb Lepore–Hollandia carriers, two were double heterozygotes for Hb Lepore/α+-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/α0 -thalassemia (SEA deletion). No α-thalassemia was detected among 4 carriers with Hb Lepore–Washington–Boston. To confirm these findings 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 δβ-hybrid Hb Lepore mutation. The 599-bp fragment generated with primers (G58 and G59) is used to specify simultaneously the Hb Lepore–Hollandia type. Using this method of DNA analysis, all 14 Hb Lepore–Hollandia carriers and 4 Hb Lepore– Washington–Boston were correctly confirmed. As Hb Lepore types have been found in diverse populations, we further established the βglobin gene haplotypes associated with the two Hb Lepore genes in these Thai patients. With this analysis, it was found that all the Thai Hb Lepore–Hollandia chromosomes were associated with an identical haplotype; (− + − + + − +) whereas the Hb Lepore–Washington– 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. βLH; β-Lepore– Hollandia, βWB; β-Washington–Boston, -SEA; α0 -thalassemia (SEA deletion), -α3.7; α+ -thalassemia (3.7 kb deletion), αCS; Hb Constant Spring. Parameters
Heterozygous Hb Lepore–Hollandia
Heterozygous Hb Lepore–Hollandia with α+-thalassemia
Heterozygous Hb Lepore–Hollandia with Hb constant spring
Heterozygous Hb Lepore–Hollandia with α0-thalassemia
Heterozygous Hb Lepore–Hollandia with Hb E
Heterozygous Hb Lepore–Washington–Boston
No β-genotype α-genotype
8 βA/βLH αα /αα
2 βA/βLH − α3.7/ αα
1 βA/βLH αCSα/ αα
1 βA/βLH −−SEA/ αα
2 βE/βLH αα / αα
4 βA/βWB αα / αα
Rbc (1012/L) Hb (g/dL) Hct (%) MCV (fL) MCH (pg) MCHC (g/dL) RDW-CV (%)
5.8 ± 0.6 12.3 ± 0.7 40.2 ± 3.7 70.2 ± 1.7 21.4 ± 0.9 30.8 ± 1.2 18.8 ± 0.4
4.4, 4.6 10.2, 12.1 32.0, 36.0 71.1, 79.0 22.9, 26.5 33.3, 34.2 16.5, 15.4
5.1 11.9 35.7 70.4 23.5 33.3 17.0
4.9 10.9 34.9 70.0 21.9 31.3 16.5
4.3, 4.0 10.3, 9.7 29.1, 28.0 68.1, 69.8 24.1, 24.2 35.4, 34.6 22.8, 22.0
5.7 ± 0.6 11.3 ± 0.6 37.9 ± 2.7 66.6 ± 3.0 19.9 ± 1.6 29.9 ± 2.0 17.1 ± 0.6
2.3 ± 0.3 4.0 ± 1.9 11.7 ± 1.0 None
2.3, 2.1 5.5, 6.2 10.8, 11.5 none
2.2 15.7 8.7 none
1.8 11.8 4.4 none
2.6, 2.2 46.5, 49.8 8.9, 6.4 42.0, 41.6
2.4 ± 0.2 3.1 ± 1.4 10.4 ± 0.2 None
+/+ (6), +/− (2)
+/−
+/−
+/+
+/+
+/− (1), −/− (3)
(− + − + + − +)
(− + − + + − +)
(− + − + + − +)
(− + − + + − +)
(− + − + + − +)
(+ − − − − + +/−) (1) (+ − − − − − +) (3)
Hb Hb Hb Hb
A2 (%)a F (%)a Lepore (%)a E (%)a
− 158 bp Gγ XmnI β-Lepore haplotype a b
b
Determined by the capillary zone electrophoresis (Sebia). Including 7 polymorphic sites: ɛ-HincII, Gγ-HindIII, Aγ-HindIII, ψβ-HincII, 3′Ψβ-HincII, β-AvaII and 3′β-BamHI.
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presented for the first 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 δ- and β-globin genes resulting in the synthesis of δβ-hybrid globin chain consisting of the N-terminal end of δ-globin and C-terminal end of β-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 Lepore–Washington–Boston has been localized to being between δ87/βIVS2#8 or δ87/β116. The breakpoints for Hb Lepore–Hallandia and Hb Lepore Baltimore have been defined as being between codon δ22/β50, and δ50/ β86, δ68/ β84 or δ59/ β86, respectively [4,5,15]. Recently, a more complex rearrangement of δ/β globin genes causing another Hb Lepore variant, the Hb Lepore– Leiden has also been reported [16]. The δβ hybrid globin chain is inefficiently synthesized because the promoter of this gene is δ in origin which lacks the CCAAT box as well as lacking the CACC box that binds the β-globin specific transcription factor, EKLF [17]. This reduced synthesis of δβ-chain is the basis for β-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 Lepore–Washington–Boston and Hb Lepore–Baltimore are most common and have a worldwide distribution in many different ethnic groups including southern European, Asian Indians and African Americans whereas Hb Lepore–Hollandia 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. Identification of Hb Lepore–Hollandia and Hb Lepore–Washington– 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 Lepore–Hollandia genes encountered in this and another study with the (δIVS I-42/βIVS I-56) breakpoint [31] differs from that described previously in most reported cases of Hb Lepore–Hollandia (δ22/βIVS1#16) [20,22,29], although the resultant δβ 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 Lepore–Hollandia in Southeast Asian populations. The same finding was observed for the Hb Lepore– Washington–Boston. We detected two different haplotypes associated with the Hb Lepore–Washington–Boston among 4 Thai individuals examined. Identification of Hb Lepore–Hollandia and Hb Lepore– Washington–Boston on different haplotypes in these Thai patients confirms the previous suggestion of a multicentric origin of this hybrid Hb variant [32,33]. Hematological findings of these Thai individuals with Hb Lepore shown in Table 1 confirm that this Hb variant exhibits a hematological picture quite similar to that of β-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 identification of Hb Lepore is the shorter retention time in HPLC of Hb Lepore, which elutes at 3.34–3.42 min whilst that of Hb A2 is 3.59–3.65 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 confirmatory 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 Lepore–Hollandia and α+-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 α+-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/α+thalassemia (-α3.7), 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/α0-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 (αCS2βA2) or Hb Constant Spring–Lepore (αCS2δβLH2) 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 α+-thalassemia, it has been proposed that when the availability of α-globin chain is decreased in α-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 Lepore–Baltimore carriers with a high Hb F and low Hb F phenotype that the XmnI (+) at the − 158 Gγ and the (AT)x(T)y repeat region at −540 bp of the β-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/α0-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 Lepore–Washington– Boston who had lower Hb F levels (3.1 ± 1.4%) as shown in Table 1. The configuration of the (AT)x(T)y repeat region at −540 bp of the βglobin gene in trans to these Lepore chromosomes remains to be elucidated. The results in Table 1 confirm that the clinical features and hematological phenotypes of the two patients with compound Hb Lepore/Hb E mimic Hb E-β-thalassemia disease commonly encountered in northeast Thailand but with relatively milder phenotype [35]. In fact, the two patients were initially diagnosed as having Hb E-βthalassemia disease. Although having hypochromic microcytic anemia, 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 confirms 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 hemoglobinopathies 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
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electrophoresis system is helpful in the initial recognition of Hb Lepore. Accurate diagnosis of this clinically relevant hemoglobinopathy 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 Office 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) 65–88. [2] S. Fucharoen, P. Winichagoon, N. Sirithanaratkul, J. Chowthaworn, P. Pootrakul, αand β- thalassemia in Thailand, Ann. N.Y. Acad. Sci 850 (1998) 412–414. [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) 143–161. [5] K.D. Lanclos, J. Patterson, G.D. Efremov, et al., Characterization of chromosomes with hybrid genes for Hb Lepore–Washington, Hb Lepore–Baltimore, Hb P-Nilotic, Hb Kenya, Hum. Genet. 77 (1987) 40–45. [6] S. Fucharoen, G. Fucharoen, W. Sriroongrueng, et al., Molecular basis of βthalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction, Hum. Genet. 84 (1989) 41–46. [7] G. Fucharoen, K. Sanchaisuriya, N. Sae-ung, S. Dangwibul, S. Fucharoen, A simplified screening strategy for thalassaemia and haemoglobin E in rural communities of Southeast Asia, Bull. World Health Organ. 82 (2004) 364–372. [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) 113–118. [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) 57–65. [10] K. Sanchaisuriya, G. Fucharoen, N. Sae-ung, A. Jetsrisuparb, S. Fucharoen, Molecular and hematologic features of hemoglobin E heterozygotes with different forms of α-thalassemia in Thailand, Ann. Hematol. 82 (2003) 612–616. [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) 149–154. [12] N. Sae-ung, G. Fucharoen, K. Sanchaisuriya, S. Fucharoen, α0-thalassemia and related disorders in northeast Thailand: a molecular and hematological characterization, Acta Haematol. 117 (2007) 78–82. [13] Y. Fukumaki, S. Fucharoen, Generation and spread of globin gene mutations in populations: β-thalassemia in Asian countries, in: M Kimaru, N Takahata (Eds.), New aspects of the genetics of molecular evolution, Springer-Verlag, Berlin, 1991, pp. 153–176.
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