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Thalassemia intermedia associated with the Hb Constant Spring EE Bart's disease in pregnancy: A molecular and hematological analysis
Blood Cells, Molecules, and Diseases 39 (2007) 195 – 198 www.elsevier.com/locate/ybcmd
Thalassemia intermedia associated with the Hb Constant Spring EE Bart's disease in pregnancy: A molecular and hematological analysis Supan Fucharoen ⁎, Goonnapa Fucharoen, Nattaya Sae-ung, Kanokwan Sanchaisuriya Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand Submitted 16 May 2007 Available online 22 June 2007 (Communicated by E. Beutler, M.D., 22 May 2007)
Abstract We defined the molecular basis and analyzed hematological phenotype associated with an unusual form of thalassemia intermedia caused by interaction of the hemoglobin Constant Spring (Hb CS), homozygous Hb E and α°-thalassemia found in two unrelated pregnant Thai women. Both patients had moderate anemia and characteristic of thalassemia intermedia. Hb-HPLC analysis demonstrated in both cases, Hb E and Hb Constant Spring with 3–4% Hb Bart's. Hb F was marginally elevated (3–5%). Both of them were diagnosed hematologically as the Hb CS EE Bart's disease. DNA analysis revealed the homozygosity for Hb E in both cases and identified the Hb CS mutation in trans to the α°thalassemia allele with the SEA deletion in one case and with the Thai deletion in another. The appearance of Hb-HPLC peak resembling the Hb CS in peripheral blood of the two cases indicated the ability to form a tetrameric Hb molecule between αCS and βE chains leading to a hybrid E CS A Hb namely the Hb E-CS (αCS 2 β2 ) with similar characteristics to Hb CS (α2 β2 ). Hematological data of the patients were presented comparatively with other forms of related disorders in our series including 2 Hb H/Hb EE diseases, 16 homozygous Hb CS with and without Hb E, 14 Hb H diseases and 35 Hb H-CS diseases. Different genotype–phenotype correlations observed in these Thai patients with these disorders are illustrated. © 2007 Elsevier Inc. All rights reserved. Keywords: Thalassemia intermedia; Hemoglobin E; Hemoglobin Constant Spring; α-Thalassemia
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 latter exceeding 50% in some minority groups of northeast Thailand. Even though most of the α-thalassemias are caused by a single (α+-thalassemia) or a double (α°-thalassemia) αglobin gene deletion, non-deletional forms of α+-thalassemia are occasionally found. The frequency of Hb Constant Spring (Hb CS), an α+-thalassemia caused by a termination codon mutation of the α2-globin gene, TAA→CAA, was found to be 1–8% [1] and a significant frequency of another α2-globin
⁎ Corresponding author. Fax: +66 43 202 083. E-mail address: [email protected] (S. Fucharoen). 1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2007.05.002
gene termination codon mutation, TAA→TAT causing the Hb Pakse′, has been found in Thai population [2,3]. Given such a carrier rate, complex interactions between them resulting in a wide spectrum of clinical syndromes are commonly encountered. Interaction of α°-thalassemia and α+-thalassemia leads to the Hb H disease and interaction of Hb H disease with Hb E heterozygote and Hb E homozygote results in complex thalassemia syndromes known as the AE Bart's and EF Bart's diseases [4–6]. We have now described the hematological and molecular characterization of an unusual condition of thalassemia intermedia caused by interactions of the Hb CS, α°thalassemia and homozygous Hb E leading to the Hb CS EE Bart's disease found in two unrelated Thai pregnant women. Different genotype–phenotype interactions of Hb CS with Hb E in our series and the demonstration of a novel hybrid Hb namely the Hb E-CS observed in the two patients are presented.
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S. Fucharoen et al. / Blood Cells, Molecules, and Diseases 39 (2007) 195–198
Materials and methods
Results and discussion
Subjects
Thalassemia intermedia encompasses a number of clinical conditions ranging in severity from a thalassemia carrier state to transfusion dependent thalassemia major. Patients with thalassemia intermedia usually have a mild anemia with base line Hb ranging from 7.0–9.0 g/dl and survive without needing regular blood transfusion. At the genotype level, thalassemia intermedia is very heterogeneous [12]. We described an unusual form of thalassemia intermedia in pregnant Thai women. Hematological parameters and genotypes of the two probands (P1 and P2) are summarized in Table 1. For comparison, data from other groups of patients with related diseases in our series [5,13] are also presented. The two probands had positive results with the thalassemia screening using osmotic fragility and DCIP tests. They were generally healthy although both of them had moderate hypochromic microcytic anemia with Hbs of 7.6 and 7.8 g/dl and MCVs of 64.7 and 64.3 fl, respectively, the characteristic features of thalassemia intermedia [12]. As shown in Fig. 1, routine Hb-HPLC analysis revealed eluting peaks of Hbs Bart's, F, Hb CS and major peak indicating homozygous Hb E in both cases. The levels of Hb F and Hb E were 3.9% and 5.7% and 81.0% and 85.7%, respectively. No Hb A and Hb H (β4) was detectable. These results are compatible with the initial phenotypic diagnosis of the Hb CS EE Bart's disease, a previously un-described phenotypic expression. Further DNA analysis identified the homozygosity for βE mutation and the αCS mutation in both cases. Gap-PCR analysis of α-globin gene identified the α°-thalassemia with the SEA deletion in P1 and with the THAI deletion in P2 in trans to the αCS mutation (Fig. 2). Therefore, the two probands were in fact the thalassemia intermedia patients that resulted from the interaction of homozygous Hb E and Hb H disease with Hb CS. This gene– gene interaction should result in the so-called Hb CS EF Bart's [6] rather than the Hb CS EE Bart's diseases. The relatively lower Hb F in our two cases indicates the genetic heterogeneity of γ-globin gene expression in these complex thalassemia syndromes. We and other investigators have previously encountered Thai patients with Hb H/Hb EE disease with
Ethical approval of the study protocol was obtained from the Ethical Committee of Khon Kaen University, Thailand (HE480830). After informed consent was obtained, EDTAanticoagulated blood specimens were collected from two pregnant Thai women attending the ongoing thalassemia screening program at the Health Promotion Center, Region 5 and Region 6 of northeast Thailand. Blood specimens were transferred to Khon Kaen University because of abnormal Hb compositions for further Hb and DNA analysis. Apart from mild anemia with hypochromic microcytosis usually associated with pregnancy in Thailand, they were in good general health and have no history of organomegaly or history of blood transfusion. Erythrocyte and Hb analysis was carried out at 20 weeks of gestation. For comparison, additional data were also obtained from 2 adult patients with Hb H/Hb EE, 3 homozygous CS/Hb EA, 1 homozygous Hb CS/Hb EA, 12 homozygous Hb CS, 14 Hb H disease and 35 Hb H-Hb CS in our series. Hemoglobin and DNA analysis Initial thalassemia screening was performed using a combined osmotic fragility (OF) and dichlorophenolindophenol (DCIP) tests described previously [7,8]. Hematological data were collected using an automated blood cell counter (Coulter STKS; Beckman Coulter Co., Fullerton, CA, USA). Hb analysis was carried out by electrophoresis on cellulose acetate and by HPLC (Variant™, Bio-Rad Laboratories, Hercules, CA, USA). Other hematological analysis was carried out using standard methods. Identifications of the Hb CS and Hb E mutations were performed using the allele specific PCR described elsewhere [9,10]. Identification of α°-thalassemia with the SEA and THAI determinants was routinely performed in our laboratory using a multiplex gap PCR described [5,11].
Table 1 Hematological data and genotypes of the two probands with Hb Constant Spring EE Bart's disease as compared to other related genotypes in our series
Number OF/DCIP Hb (g/dl) Hct (%) MCV (fl) MCH (pg) MCHC (g/dl) RDW (%) Hb A2/E (%) Hb F (%) Hb types α-genotype β-genotype
P1
P2
Hb H/Hb EE
Homozygous Hb CS/Hb EE*
Homozygous Hb CS/Hb EA*
Homozygous Hb CS*
Hb H**
Hb H-CS**
1 +/+ 7.6 32.8 64.7 15.0 23.2 23.2 81.0 3.9 CS EE Bart's αCSα/–SEA βE/βE
1 +/+ 7.8 34.5 64.3 15.4 23.1 24.6 85.7 5.7 CS EE Bart's αCSα/–THAI βE/βE
2 +/+ 12.2, 11.5 40.6, 33.0 54.2, 45.6 16.3, 15.9 30.0, 34.8 17.0, 21.9 87.2, 89.5 3.1, 2.0 EE − α3.7/–SEA βE/βE
3 +/+ 11.5 ± 0.7 36.5 ± 1.5 70.0 ± 5.2 22.0 ± 0.7 31.6 ± 2.6 16.9 ± 2.1 87.6 ± 2.4 2.0 ± 0.4 CS EE αCSα/αCSα βE/βE
1 +/+ 7.9 27.6 70.1 20.1 28.5 28.1 23.9 0.5 CS E A αCSα/αCSα βA/βE
12 +/− 10.1 ± 1.0 32.6 ± 3.3 75.6 ± 6.2 23.3 ± 2.0 30.7 ± 0.6 17.8 ± 4.0 2.2 ± 0.6 0.8 ± 0.9 CS A2A Bart's αCSα/αCSα βA/βA
14 +/− 8.9 +/+ 1.1 30.1 ± 4.0 60.7 ± 7.1 17.6 ± 1.5 29.1 ± 1.7 24.5 ± 3.1 1.1 ± 0.3 1.6 ± 0.9 A2A Bart's H − α3.7/–SEA βA/βA
35 +/− 8.2 ± 1.0 30.4 ± 3.8 73.5 ± 6.8 19.8 ± 1.7 27.0 ± 1.9 23.9 ± 4.9 1.3 ± 1.3 2.3 ± 2.2 CS A2A Bart's H αCSα/–SEA βA/βA
Values are presented as raw data or as mean ± SD where appropriate. Data from *Singsanan S, et al. (2007) [13] and **Boonsa S, et al. (2004) [5].
S. Fucharoen et al. / Blood Cells, Molecules, and Diseases 39 (2007) 195–198
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Fig. 1. Hb-HPLC analysis of the homozygous Hb CS, homozygous Hb CS/Hb EE and the two probands (P1 and P2) with Hb CS EE Bart's disease indicating separating peaks of Hbs Bart's, F, A, A2, CS, E and the hybrid Hb E-CS.
unusually lower productions of Hb F and Hb Bart's and pitfall in clinical diagnosis and genetic counseling of the diseases [14– 16]. Although with different α°-thalassemia alleles (SEA and THAI), the two probands had very similar hematological parameters, confirming the same phenotypic expression of the two α°-thalassemia determinants [11,17]. Based on these findings, it is conceivable that the Hb CS peaks observed on
the Hb-HPLC analysis of the peripheral blood of the two probands are in fact the hybrid Hb molecule resulting from a tetrameric assembly of two αCS and two βE chains, namely the Hb E-CS (α2CSβ2E) rather than the Hb CS with two αCS and two βA chains (α2CSβ2A) as there is no βA chain in these two patients. This hybrid Hb E-CS was also observed in 3 other cases of Thai patients with homozygous Hb CS/Hb EE although no Hb Bart's
Fig. 2. Multiplex allele specific PCR for simultaneous detection of the SEA and the THAI deletional α°-thalassemia. P1 and P2 are carriers of α°-thalassemia with the SEA and the THAI deletion, respectively. M is λ/HindIII size markers.
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was detectable in these latter cases as shown in Fig. 1. This hybrid Hb migrated to the same position with that of the Hb CS on cellulose acetate electrophoresis in alkaline pH (data not shown). It is also noteworthy from the eluting peak and the amount observed on the Hb-HPLC analysis that this hybrid Hb has very similar chromatographic, electrophoretic and stability properties to that of the Hb CS. We have also reported previously similar hybrid Hb molecules between βE chain and other α-globin variants leading to the Hb QE (α2Qβ2E) and Hb E Beijing (α2BJβ2E) in Thai families with thalassemia intermedia phenotypes [18,19]. This observation of the hybrid Hb E-CS in our cases provides an in vivo evidence for the ability of the αCS chain to form a tetrameric Hb molecule with the βE chain. As shown in Table 1, when the hematological data of the two patients (P1 and P2) with the Hb CS EE Bart's disease were compared with those of the 2 cases of Hb H/Hb EE disease and 3 cases of the homozygous Hb CS/Hb EE, it is apparent that the Hb CS EE Bart's disease is associated with a more severe phenotype with lower Hb and higher RDW values. Moreover, the presence of Hb Bart's (γ4) in the peripheral blood of the two probands but not in the latter two syndromes indicates likely a more γ-globin excess, due primarily to a more limited availability of α-globin chain for this Hb CS EE Bart's disease as compared to the Hb H/Hb EE and the homozygous Hb CS/Hb EE. It is also noteworthy when the hematological data of the two probands were compared with those of 14 Hb H and 35 Hb HCS diseases that co-inheritance of homozygous Hb E (as observed for the two probands) could not ameliorate the severity of Hb H and Hb H-CS diseases in our patients as usually observed for other common forms of thalassemia [20]. However, too few cases with the same genotype had been documented before for comparison of the hematological phenotypes. Nonetheless, identification of our Thai patients with the unusual form of thalassemia intermedia confirms that for areas where both thalassemia and hemoglobinopathies are prevalent as in Southeast Asia, complex syndromes may result from the interaction of several defects with a spectrum of clinical and hematological manifestations. It is therefore important to understand these gene–gene interactions to be able to provide better prediction of the clinical outcome of the patients and improving genetic counseling. Acknowledgments This work was supported by a Research Grant from Khon Kaen University, Khon Kaen, Thailand. We thank Ms. Yupin Jopang and Mrs. Mathanee Chiwasatienchai of the Health Promotion Center, Region 5 and Region 6 of the Ministry of Public Health, Thailand for help with recruitment of the patients and specimens collection. References [1] S. Fucharoen, P. Winichagoon, N. Sirithanaratkul, J. Chowthaworn, P. Pootrakul, α and β-thalassemia in Thailand, Ann. N. Y. Acad. Sci. 850 (1998) 412–414.
[2] K. Sanchaisuriya, G. Fucharoen, S. Fucharoen, Hb Paksé [α2, codon 142 (TAA-NTAT or Term-NTyr)] in Thai patients with EABart's disease and Hb H disease, Hemoglobin 26 (2002) 227–235. [3] V. Viprakasit, V.S. Tanphaichitr, P. Pung-Amritt, S. Petrarat, L. Suwantol, C. Fisher, D.R. Higgs, Clinical phenotypes and molecular characterization of Hb H-Paksé disease, Haematologica 87 (2002) 117–125. [4] S. Fucharoen, P. Winichagoon, W. Prayoonwiwat, P. Pootrakul, A. Piankitjagum, P. Wasi, Clinical and hematologic manifestations of AEBart's disease, Birth Defects Orig. Artic. Ser. 23/5A (1988) 327–332. [5] S. Boonsa, K. Sanchaisuriya, G. Fucharoen, S. Wiangnon, A. Jetsrisuparb, S. Fucharoen, The diverse molecular basis and hematological features of Hb H and AEBart's diseases in northeast Thailand, Acta Haematol. 111 (2004) 149–154. [6] S. Fucharoen, P. Winichagoon, V. Thonglairua, P. Wasi, EFBart's disease: interaction of the abnormal α- and β-globin genes, Eur. J. Haematol. 40 (1988) 75–78. [7] G. Fucharoen, K. Sanchaisuriya, N. Sae-ung, S. Dangwibul, S. Fucharoen, A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia, Bull. World Health Organ. 82 (2004) 364–372. [8] K. Sanchaisuriya, S. Fucharoen, G. Fucharoen, T. Ratanasiri, P. Sanchaisuriya, Y. Changtrakul, U. Ukosanakarn, W. Ussawaphark, F.P. Schelp, 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] K. Sanchaisuriya, G. Fucharoen, N. Sae-ung, A. Jetsrisuparb, S. Fucharoen, Molecular and hematologic features hemoglobin E heterozygotes with different forms of α-thalassemia in Thailand, Ann. Hematol. 82 (2003) 612–616. [10] S. Fucharoen, K. Sanchaisuriya, G. Fucharoen, S. Panyasai, R. Devenish, L. Luy, Interaction of hemoglobin E and several forms of α-thalassemia in Cambodian families, Haematologica 88 (2003) 1092–1098. [11] N. Sae-ung, G. Fucharoen, K. Sanchaisuriya, S. Fucharoen, α°Thalassemia and related disorders in northeast Thailand: a molecular and hematological characterization, Acta Haematol. 117 (2007) 78–82. [12] R. Galanello, A. Cao, Relationship between genotype and phenotype; thalassemia intermedia, Ann. N. Y. Acad. Sci. 850 (1998) 325–333. [13] S. Singsanan, G. Fucharoen, K. Sanchaisuriya, O. Savongsy, S. Fucharoen, Molecular characterization and origins of Hb Constant Spring and Hb Paksé in Southeast Asian population. Ann Hematol (in press). [14] G. Fucharoen, J. Trithipsombat, S. Sirithawee, S. Yamsri, Y. Changtrakul, K. Sanchaisuriya, S. Fucharoen, Molecular and hematological profiles of hemoglobin EE disease with different forms of α-thalassemia, Ann. Hematol. 85 (2006) 450–454. [15] V. Viprakasit, V.S. Tanphaichitr, Unusual phenotype of hemoglobin EE with hemoglobin H disease: a pitfall in clinical diagnosis and genetic counseling, J. Pediatr. 144 (2004) 391–393. [16] S.H. Emburry, G.L. Kropp, T.S. Stanton, T.C. Warren, P.A. Cornett, F.F. Chehab, Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping, Blood 76 (1990) 619–623. [17] N. Siriratmanawong, C. Pinmuang-ngam, G. Fucharoen, S. Fucharoen, Prenatal diagnosis of Hb Bart's hydrops fetalis caused by a genetic compound heterozygosity for two different α°-thalassemia determinants, Fetal Diagn. Ther. 22 (2007) 264–268. [18] K. Sanchaisuriya, S. Chunpanich, S. Fucharoen, G. Fucharoen, P. Sanchaisuriya, Y. Changtrakun, Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy, Eur. J. Haematol. 74 (2005) 221–227. [19] S. Fucharoen, S. Chunpanich, K. Sanchaisuriya, G. Fucharoen, N. Kunyanone, Thalassemia intermedia associated with complex interaction of Hb Beijing [α16(A14)Lys–Asn] and Hb E [β26(B8)Glu–Lys] with a deletional α-thalassemia 1 in a Thai family, Hemoglobin 29 (2005) 77–83. [20] D.J. Weatherall, The molecular basis for phenotypic variability of the common thalasssemias, Mol. Med. Today 1 (1995) 15–20.
Thalassemia intermedia associated with the Hb Constant Spring EE Bart's disease in pregnancy: A molecular and hematological analysis Supan Fucharoen ⁎, Goonnapa Fucharoen, Nattaya Sae-ung, Kanokwan Sanchaisuriya Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand Submitted 16 May 2007 Available online 22 June 2007 (Communicated by E. Beutler, M.D., 22 May 2007)
Abstract We defined the molecular basis and analyzed hematological phenotype associated with an unusual form of thalassemia intermedia caused by interaction of the hemoglobin Constant Spring (Hb CS), homozygous Hb E and α°-thalassemia found in two unrelated pregnant Thai women. Both patients had moderate anemia and characteristic of thalassemia intermedia. Hb-HPLC analysis demonstrated in both cases, Hb E and Hb Constant Spring with 3–4% Hb Bart's. Hb F was marginally elevated (3–5%). Both of them were diagnosed hematologically as the Hb CS EE Bart's disease. DNA analysis revealed the homozygosity for Hb E in both cases and identified the Hb CS mutation in trans to the α°thalassemia allele with the SEA deletion in one case and with the Thai deletion in another. The appearance of Hb-HPLC peak resembling the Hb CS in peripheral blood of the two cases indicated the ability to form a tetrameric Hb molecule between αCS and βE chains leading to a hybrid E CS A Hb namely the Hb E-CS (αCS 2 β2 ) with similar characteristics to Hb CS (α2 β2 ). Hematological data of the patients were presented comparatively with other forms of related disorders in our series including 2 Hb H/Hb EE diseases, 16 homozygous Hb CS with and without Hb E, 14 Hb H diseases and 35 Hb H-CS diseases. Different genotype–phenotype correlations observed in these Thai patients with these disorders are illustrated. © 2007 Elsevier Inc. All rights reserved. Keywords: Thalassemia intermedia; Hemoglobin E; Hemoglobin Constant Spring; α-Thalassemia
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 latter exceeding 50% in some minority groups of northeast Thailand. Even though most of the α-thalassemias are caused by a single (α+-thalassemia) or a double (α°-thalassemia) αglobin gene deletion, non-deletional forms of α+-thalassemia are occasionally found. The frequency of Hb Constant Spring (Hb CS), an α+-thalassemia caused by a termination codon mutation of the α2-globin gene, TAA→CAA, was found to be 1–8% [1] and a significant frequency of another α2-globin
⁎ Corresponding author. Fax: +66 43 202 083. E-mail address: [email protected] (S. Fucharoen). 1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2007.05.002
gene termination codon mutation, TAA→TAT causing the Hb Pakse′, has been found in Thai population [2,3]. Given such a carrier rate, complex interactions between them resulting in a wide spectrum of clinical syndromes are commonly encountered. Interaction of α°-thalassemia and α+-thalassemia leads to the Hb H disease and interaction of Hb H disease with Hb E heterozygote and Hb E homozygote results in complex thalassemia syndromes known as the AE Bart's and EF Bart's diseases [4–6]. We have now described the hematological and molecular characterization of an unusual condition of thalassemia intermedia caused by interactions of the Hb CS, α°thalassemia and homozygous Hb E leading to the Hb CS EE Bart's disease found in two unrelated Thai pregnant women. Different genotype–phenotype interactions of Hb CS with Hb E in our series and the demonstration of a novel hybrid Hb namely the Hb E-CS observed in the two patients are presented.
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S. Fucharoen et al. / Blood Cells, Molecules, and Diseases 39 (2007) 195–198
Materials and methods
Results and discussion
Subjects
Thalassemia intermedia encompasses a number of clinical conditions ranging in severity from a thalassemia carrier state to transfusion dependent thalassemia major. Patients with thalassemia intermedia usually have a mild anemia with base line Hb ranging from 7.0–9.0 g/dl and survive without needing regular blood transfusion. At the genotype level, thalassemia intermedia is very heterogeneous [12]. We described an unusual form of thalassemia intermedia in pregnant Thai women. Hematological parameters and genotypes of the two probands (P1 and P2) are summarized in Table 1. For comparison, data from other groups of patients with related diseases in our series [5,13] are also presented. The two probands had positive results with the thalassemia screening using osmotic fragility and DCIP tests. They were generally healthy although both of them had moderate hypochromic microcytic anemia with Hbs of 7.6 and 7.8 g/dl and MCVs of 64.7 and 64.3 fl, respectively, the characteristic features of thalassemia intermedia [12]. As shown in Fig. 1, routine Hb-HPLC analysis revealed eluting peaks of Hbs Bart's, F, Hb CS and major peak indicating homozygous Hb E in both cases. The levels of Hb F and Hb E were 3.9% and 5.7% and 81.0% and 85.7%, respectively. No Hb A and Hb H (β4) was detectable. These results are compatible with the initial phenotypic diagnosis of the Hb CS EE Bart's disease, a previously un-described phenotypic expression. Further DNA analysis identified the homozygosity for βE mutation and the αCS mutation in both cases. Gap-PCR analysis of α-globin gene identified the α°-thalassemia with the SEA deletion in P1 and with the THAI deletion in P2 in trans to the αCS mutation (Fig. 2). Therefore, the two probands were in fact the thalassemia intermedia patients that resulted from the interaction of homozygous Hb E and Hb H disease with Hb CS. This gene– gene interaction should result in the so-called Hb CS EF Bart's [6] rather than the Hb CS EE Bart's diseases. The relatively lower Hb F in our two cases indicates the genetic heterogeneity of γ-globin gene expression in these complex thalassemia syndromes. We and other investigators have previously encountered Thai patients with Hb H/Hb EE disease with
Ethical approval of the study protocol was obtained from the Ethical Committee of Khon Kaen University, Thailand (HE480830). After informed consent was obtained, EDTAanticoagulated blood specimens were collected from two pregnant Thai women attending the ongoing thalassemia screening program at the Health Promotion Center, Region 5 and Region 6 of northeast Thailand. Blood specimens were transferred to Khon Kaen University because of abnormal Hb compositions for further Hb and DNA analysis. Apart from mild anemia with hypochromic microcytosis usually associated with pregnancy in Thailand, they were in good general health and have no history of organomegaly or history of blood transfusion. Erythrocyte and Hb analysis was carried out at 20 weeks of gestation. For comparison, additional data were also obtained from 2 adult patients with Hb H/Hb EE, 3 homozygous CS/Hb EA, 1 homozygous Hb CS/Hb EA, 12 homozygous Hb CS, 14 Hb H disease and 35 Hb H-Hb CS in our series. Hemoglobin and DNA analysis Initial thalassemia screening was performed using a combined osmotic fragility (OF) and dichlorophenolindophenol (DCIP) tests described previously [7,8]. Hematological data were collected using an automated blood cell counter (Coulter STKS; Beckman Coulter Co., Fullerton, CA, USA). Hb analysis was carried out by electrophoresis on cellulose acetate and by HPLC (Variant™, Bio-Rad Laboratories, Hercules, CA, USA). Other hematological analysis was carried out using standard methods. Identifications of the Hb CS and Hb E mutations were performed using the allele specific PCR described elsewhere [9,10]. Identification of α°-thalassemia with the SEA and THAI determinants was routinely performed in our laboratory using a multiplex gap PCR described [5,11].
Table 1 Hematological data and genotypes of the two probands with Hb Constant Spring EE Bart's disease as compared to other related genotypes in our series
Number OF/DCIP Hb (g/dl) Hct (%) MCV (fl) MCH (pg) MCHC (g/dl) RDW (%) Hb A2/E (%) Hb F (%) Hb types α-genotype β-genotype
P1
P2
Hb H/Hb EE
Homozygous Hb CS/Hb EE*
Homozygous Hb CS/Hb EA*
Homozygous Hb CS*
Hb H**
Hb H-CS**
1 +/+ 7.6 32.8 64.7 15.0 23.2 23.2 81.0 3.9 CS EE Bart's αCSα/–SEA βE/βE
1 +/+ 7.8 34.5 64.3 15.4 23.1 24.6 85.7 5.7 CS EE Bart's αCSα/–THAI βE/βE
2 +/+ 12.2, 11.5 40.6, 33.0 54.2, 45.6 16.3, 15.9 30.0, 34.8 17.0, 21.9 87.2, 89.5 3.1, 2.0 EE − α3.7/–SEA βE/βE
3 +/+ 11.5 ± 0.7 36.5 ± 1.5 70.0 ± 5.2 22.0 ± 0.7 31.6 ± 2.6 16.9 ± 2.1 87.6 ± 2.4 2.0 ± 0.4 CS EE αCSα/αCSα βE/βE
1 +/+ 7.9 27.6 70.1 20.1 28.5 28.1 23.9 0.5 CS E A αCSα/αCSα βA/βE
12 +/− 10.1 ± 1.0 32.6 ± 3.3 75.6 ± 6.2 23.3 ± 2.0 30.7 ± 0.6 17.8 ± 4.0 2.2 ± 0.6 0.8 ± 0.9 CS A2A Bart's αCSα/αCSα βA/βA
14 +/− 8.9 +/+ 1.1 30.1 ± 4.0 60.7 ± 7.1 17.6 ± 1.5 29.1 ± 1.7 24.5 ± 3.1 1.1 ± 0.3 1.6 ± 0.9 A2A Bart's H − α3.7/–SEA βA/βA
35 +/− 8.2 ± 1.0 30.4 ± 3.8 73.5 ± 6.8 19.8 ± 1.7 27.0 ± 1.9 23.9 ± 4.9 1.3 ± 1.3 2.3 ± 2.2 CS A2A Bart's H αCSα/–SEA βA/βA
Values are presented as raw data or as mean ± SD where appropriate. Data from *Singsanan S, et al. (2007) [13] and **Boonsa S, et al. (2004) [5].
S. Fucharoen et al. / Blood Cells, Molecules, and Diseases 39 (2007) 195–198
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Fig. 1. Hb-HPLC analysis of the homozygous Hb CS, homozygous Hb CS/Hb EE and the two probands (P1 and P2) with Hb CS EE Bart's disease indicating separating peaks of Hbs Bart's, F, A, A2, CS, E and the hybrid Hb E-CS.
unusually lower productions of Hb F and Hb Bart's and pitfall in clinical diagnosis and genetic counseling of the diseases [14– 16]. Although with different α°-thalassemia alleles (SEA and THAI), the two probands had very similar hematological parameters, confirming the same phenotypic expression of the two α°-thalassemia determinants [11,17]. Based on these findings, it is conceivable that the Hb CS peaks observed on
the Hb-HPLC analysis of the peripheral blood of the two probands are in fact the hybrid Hb molecule resulting from a tetrameric assembly of two αCS and two βE chains, namely the Hb E-CS (α2CSβ2E) rather than the Hb CS with two αCS and two βA chains (α2CSβ2A) as there is no βA chain in these two patients. This hybrid Hb E-CS was also observed in 3 other cases of Thai patients with homozygous Hb CS/Hb EE although no Hb Bart's
Fig. 2. Multiplex allele specific PCR for simultaneous detection of the SEA and the THAI deletional α°-thalassemia. P1 and P2 are carriers of α°-thalassemia with the SEA and the THAI deletion, respectively. M is λ/HindIII size markers.
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was detectable in these latter cases as shown in Fig. 1. This hybrid Hb migrated to the same position with that of the Hb CS on cellulose acetate electrophoresis in alkaline pH (data not shown). It is also noteworthy from the eluting peak and the amount observed on the Hb-HPLC analysis that this hybrid Hb has very similar chromatographic, electrophoretic and stability properties to that of the Hb CS. We have also reported previously similar hybrid Hb molecules between βE chain and other α-globin variants leading to the Hb QE (α2Qβ2E) and Hb E Beijing (α2BJβ2E) in Thai families with thalassemia intermedia phenotypes [18,19]. This observation of the hybrid Hb E-CS in our cases provides an in vivo evidence for the ability of the αCS chain to form a tetrameric Hb molecule with the βE chain. As shown in Table 1, when the hematological data of the two patients (P1 and P2) with the Hb CS EE Bart's disease were compared with those of the 2 cases of Hb H/Hb EE disease and 3 cases of the homozygous Hb CS/Hb EE, it is apparent that the Hb CS EE Bart's disease is associated with a more severe phenotype with lower Hb and higher RDW values. Moreover, the presence of Hb Bart's (γ4) in the peripheral blood of the two probands but not in the latter two syndromes indicates likely a more γ-globin excess, due primarily to a more limited availability of α-globin chain for this Hb CS EE Bart's disease as compared to the Hb H/Hb EE and the homozygous Hb CS/Hb EE. It is also noteworthy when the hematological data of the two probands were compared with those of 14 Hb H and 35 Hb HCS diseases that co-inheritance of homozygous Hb E (as observed for the two probands) could not ameliorate the severity of Hb H and Hb H-CS diseases in our patients as usually observed for other common forms of thalassemia [20]. However, too few cases with the same genotype had been documented before for comparison of the hematological phenotypes. Nonetheless, identification of our Thai patients with the unusual form of thalassemia intermedia confirms that for areas where both thalassemia and hemoglobinopathies are prevalent as in Southeast Asia, complex syndromes may result from the interaction of several defects with a spectrum of clinical and hematological manifestations. It is therefore important to understand these gene–gene interactions to be able to provide better prediction of the clinical outcome of the patients and improving genetic counseling. Acknowledgments This work was supported by a Research Grant from Khon Kaen University, Khon Kaen, Thailand. We thank Ms. Yupin Jopang and Mrs. Mathanee Chiwasatienchai of the Health Promotion Center, Region 5 and Region 6 of the Ministry of Public Health, Thailand for help with recruitment of the patients and specimens collection. References [1] S. Fucharoen, P. Winichagoon, N. Sirithanaratkul, J. Chowthaworn, P. Pootrakul, α and β-thalassemia in Thailand, Ann. N. Y. Acad. Sci. 850 (1998) 412–414.
[2] K. Sanchaisuriya, G. Fucharoen, S. Fucharoen, Hb Paksé [α2, codon 142 (TAA-NTAT or Term-NTyr)] in Thai patients with EABart's disease and Hb H disease, Hemoglobin 26 (2002) 227–235. [3] V. Viprakasit, V.S. Tanphaichitr, P. Pung-Amritt, S. Petrarat, L. Suwantol, C. Fisher, D.R. Higgs, Clinical phenotypes and molecular characterization of Hb H-Paksé disease, Haematologica 87 (2002) 117–125. [4] S. Fucharoen, P. Winichagoon, W. Prayoonwiwat, P. Pootrakul, A. Piankitjagum, P. Wasi, Clinical and hematologic manifestations of AEBart's disease, Birth Defects Orig. Artic. Ser. 23/5A (1988) 327–332. [5] S. Boonsa, K. Sanchaisuriya, G. Fucharoen, S. Wiangnon, A. Jetsrisuparb, S. Fucharoen, The diverse molecular basis and hematological features of Hb H and AEBart's diseases in northeast Thailand, Acta Haematol. 111 (2004) 149–154. [6] S. Fucharoen, P. Winichagoon, V. Thonglairua, P. Wasi, EFBart's disease: interaction of the abnormal α- and β-globin genes, Eur. J. Haematol. 40 (1988) 75–78. [7] G. Fucharoen, K. Sanchaisuriya, N. Sae-ung, S. Dangwibul, S. Fucharoen, A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia, Bull. World Health Organ. 82 (2004) 364–372. [8] K. Sanchaisuriya, S. Fucharoen, G. Fucharoen, T. Ratanasiri, P. Sanchaisuriya, Y. Changtrakul, U. Ukosanakarn, W. Ussawaphark, F.P. Schelp, 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] K. Sanchaisuriya, G. Fucharoen, N. Sae-ung, A. Jetsrisuparb, S. Fucharoen, Molecular and hematologic features hemoglobin E heterozygotes with different forms of α-thalassemia in Thailand, Ann. Hematol. 82 (2003) 612–616. [10] S. Fucharoen, K. Sanchaisuriya, G. Fucharoen, S. Panyasai, R. Devenish, L. Luy, Interaction of hemoglobin E and several forms of α-thalassemia in Cambodian families, Haematologica 88 (2003) 1092–1098. [11] N. Sae-ung, G. Fucharoen, K. Sanchaisuriya, S. Fucharoen, α°Thalassemia and related disorders in northeast Thailand: a molecular and hematological characterization, Acta Haematol. 117 (2007) 78–82. [12] R. Galanello, A. Cao, Relationship between genotype and phenotype; thalassemia intermedia, Ann. N. Y. Acad. Sci. 850 (1998) 325–333. [13] S. Singsanan, G. Fucharoen, K. Sanchaisuriya, O. Savongsy, S. Fucharoen, Molecular characterization and origins of Hb Constant Spring and Hb Paksé in Southeast Asian population. Ann Hematol (in press). [14] G. Fucharoen, J. Trithipsombat, S. Sirithawee, S. Yamsri, Y. Changtrakul, K. Sanchaisuriya, S. Fucharoen, Molecular and hematological profiles of hemoglobin EE disease with different forms of α-thalassemia, Ann. Hematol. 85 (2006) 450–454. [15] V. Viprakasit, V.S. Tanphaichitr, Unusual phenotype of hemoglobin EE with hemoglobin H disease: a pitfall in clinical diagnosis and genetic counseling, J. Pediatr. 144 (2004) 391–393. [16] S.H. Emburry, G.L. Kropp, T.S. Stanton, T.C. Warren, P.A. Cornett, F.F. Chehab, Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping, Blood 76 (1990) 619–623. [17] N. Siriratmanawong, C. Pinmuang-ngam, G. Fucharoen, S. Fucharoen, Prenatal diagnosis of Hb Bart's hydrops fetalis caused by a genetic compound heterozygosity for two different α°-thalassemia determinants, Fetal Diagn. Ther. 22 (2007) 264–268. [18] K. Sanchaisuriya, S. Chunpanich, S. Fucharoen, G. Fucharoen, P. Sanchaisuriya, Y. Changtrakun, Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy, Eur. J. Haematol. 74 (2005) 221–227. [19] S. Fucharoen, S. Chunpanich, K. Sanchaisuriya, G. Fucharoen, N. Kunyanone, Thalassemia intermedia associated with complex interaction of Hb Beijing [α16(A14)Lys–Asn] and Hb E [β26(B8)Glu–Lys] with a deletional α-thalassemia 1 in a Thai family, Hemoglobin 29 (2005) 77–83. [20] D.J. Weatherall, The molecular basis for phenotypic variability of the common thalasssemias, Mol. Med. Today 1 (1995) 15–20.