Date Published: September 19, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Supachai Ekwattanakit, Yuwarat Monteerarat, Suchada Riolueang, Kalaya Tachavanich, Vip Viprakasit.
Background and Objectives. To explore the role of cis-regulatory sequences within the β globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with β globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the β globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different β globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a) (III), (b) (V), and (c) (IV) accounting for 94% of Hb E chromosomes. A new haplotype (Th-1) was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F (average 10.5%, range 5.8–14.3%). No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E.
Beside producing abnormal variant, hemoglobin E (HbE), the G→A substitution in codon 26 (Glu→Lys) of the β-globin gene (βE) could also produce β+ thalassemia due to decreased functional HbE-mRNA, secondary to alternative splicing mechanism . However, the clinical phenotype in homozygous Hb E (Hb EE) is rather asymptomatic with very mild anemia. In contrast, patients with HbE/β thalassemia have a more diverse clinical phenotype from transfusion dependent to very mild disease [2–5]. Although, understanding of clinical phenotypic diversity in patients with Hb E/β thalassemia has long been a topic of several investigations, at present, the genotype-phenotype correlation of this so-called single gene disorder remains obscure.
Ethylene diamine tetraacetic acid (EDTA) blood was obtained from 80 Thai individuals with homozygous HbE after informed consent. They were parents, siblings, and unrelated individuals who were tested due to microcytic anemia at Department of Pediatrics, Faculty of Medicine Siriraj Hospital as a part of an ongoing project studying natural history and genotype-phenotype correlation in Hb E disorders in Thailand. This study was approved by a local ethical committee at Mahidol University. Complete hematological and hemoglobin studies have been performed using standard techniques as described previously . Quantification of Hb E and Hb F % was performed by calculation from cellulose acetate electrophoresis and for Hb F, it was subsequently confirmed by alkaline denaturation method .
From 80 individuals with homozygous Hb E, we could successfully genotypes all 8 polymorphic sites in 77 cases (154 chromosomes of Hb E, Table 1). These individuals were mainly from Bangkok and the Central plain of Thailand. Eight different β globin haplotypes associated with HbE alleles have been constructed with two novel ones, (− − − − − + −) and (+ + − + + + −). Three major haplotypes based on Orkin et al.  III (60.4%), V (24.0%), and IV (9.7%) were accounted for the majority of HbE alleles and they were linked with Xmn I +, −, and +, respectively. Therefore only 71.43% of HbE allele is linked with the T allele (or + genotype) of Xmn I. Only the frameworks 2 (+ −) and 3 (− +) based on Antonarakis et al.  were found, while the framework associated with (−−) or (+ +) patterns were not observed. The summary of all eight β globin haplotypes is shown in Table 1.
To the best of our knowledge, this study, herein, provides the most extensive analyses of the β globin haplotypes linked to Hb E alleles to date. Due to a higher number of individuals with homozygous Hb E used in this study, we found that the haplotypes linked to Hb E in Thailand might be more heterogeneous than was previously thought. At least 8 different haplotypes including two novel ones were found in this study; however, in the majority of these cases (94.3%), Hb E alleles were linked with the three major haplotypes; (a) (III), (b) (V), and (c) (IV), consisting with previous studies in our population and supporting the hypothesis of multiple origins of Hb E in this part of the world [11–13]. In Bangkok, where it is a melting pot of the whole country due to a massive immigration of people from different parts of Thailand, it is not simple to trace and track back their original regions. Therefore it is not surprising that we found such a heterogeneity including two new haplotypes in Thailand: Th1; (− − − − − + −) and Arab-Indian; (+ + − + + + −). The latter is identical to βS haplotype reported from previous studies [20, 24]. However, these two new haplotypes were closely similar to the two common haplotypes of b (V) and a (III), respectively. Only the specific restriction site at the ε-Hind II site is different suggesting that these two novel haplotypes may arise from interallelic gene conversion than an independent origin . Moreover our study also confirms the previous construction of the β globin haplotypes in other studies which had a limited number of homozygous condition and used the haplotype analyses mainly from Hb E heterozygotes [11, 12].