Variability of hemoglobin F expression in hemoglobin EE disease: Hematological and molecular analysis

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Abstract

Although the molecular basis of variability of hemoglobin (Hb) F has been extensively examined in β-thalassemia and sickle cell diseases, less study has been done on Hb E disorder. To address the variability of Hb F expression in Hb EE disease, we have examined multiple single nucleotide polymorphisms (SNPs) in the β-globin gene cluster, BCL11A and HBS1L-MYB genes and determined their associations with Hb F levels in this syndrome. Study was done on 141 adult Thai individuals with homozygous Hb E. Hematological parameters were recorded and Hb F measured using Hb-HPLC analyzer. It was found in 26 cases that co-inheritance of α-thalassemia could lead to significant lower production of Hb F. Association of Hb F expression with the Gγ-Xmn I polymorphism and other SNPs including rs2297339, rs2838513, rs4895441 and rs9399137 in HBS1L-MYB gene and rs4671393 and rs11886868 in BCL11A gene was therefore analyzed in the remaining 115 cases without α-thalassemia. It was found that 4 of these 7 SNPs including Gγ-XmnI polymorphism (rs7482144), HBS1L-MYB (rs4895441) and (rs9399137) and BCL11A (rs4671393) were significantly associated with higher proportions of subjects with high Hb F (Hb F  5%). The result demonstrated that multiple genetic modifying factors including T allele of Gγ-XmnI polymorphism (rs7482144), G allele of HBS1L-MYB (rs489441), C allele of HBS1L-MYB (rs9399137) and C allele of BCL11A (rs4671393) are associated with increased Hb F and in combination could explain approximately 80% of the variation of Hb F in Hb EE disease in Thai population. Other genetic factors regulating Hb F expression in this common genetic disorder remains to be elucidated.

Introduction

Hemoglobin (Hb) E is the most common β-globin chain variant (α2β226Glu→Lys) in Southeast Asian populations [1]. The βE chain is synthesized at a reduced rate compared with the βA chain because the mutation creates an abnormal splicing within the exon 1 of β-globin gene [2]. While heterozygous state of Hb E is an asymptomatic condition, Hb E homozygote or Hb EE disease is usually associated with a mild hypochromic microcytosis and variability of Hb F level; ranging from 0% to 10%. Higher expression of Hb F in the Hb E syndrome is prone to a mis-diagnosis of Hb E-β-thalassemia disease, a common β-thalassemia syndrome in the region [3], [4]. The basis for this variability of Hb F in homozygous Hb E is poorly understood. However, it has been shown that the level of Hb F is regulated by a complex mechanism involving at least three major loci: the HBG2:g.-158C > T on 11p15.4 namely the Gγ-Xmn I polymorphism, the HBS1L-MYB intergenic region on 6q23.3 and the BCL11A on 2p16.1. Multiple DNA polymorphisms on these three loci have been found to be associated with high Hb F expression in the patients with β-thalassemia and sickle cell diseases as well as in healthy subjects in various populations [5], [6], [7], [8], [9], [10], [11], [12]. In this study, we have investigated these SNPs in Hb EE syndrome in northeast Thailand and determined their associations with phenotypic expression of Hb F.

Section snippets

Subjects

Peripheral blood samples of 141 adult subjects with homozygous Hb E encountered at our ongoing thalassemia screening unit at the Centre for Research and Development of Medical Diagnostic Laboratories, Khon Kaen University, Thailand were recruited in this study. Ethical approval of the study protocol was obtained from the institutional review board (IRB) of Khon Kaen University, Khon Kaen, Thailand (HE451007). Informed consent was obtained.

Hematological analysis

Screening for thalassemia and hemoglobinopathies was

Results

DNA analysis by an allele-specific PCR for βE-globin gene revealed homozygosity for the βE mutation (β26, GAG-AAG) in all 141 cases. A total of 7 SNPs in three major loci including the rs7482144 of Gγ-XmnI, 4 SNPs in HBS1L-MYB (rs2297339, rs2838513, rs4895441 and rs9399137) and 2 SNPs in BCL11A (rs4671393 and rs11886868) were examined. Table 1 lists the major and minor alleles found for these 7 SNPs and χ2 test indicated in 6 out of 7 SNPs, excluding the HBS1L-MYB (rs9399137), the assumption of

Discussion

Hb E [β26(B8)Glu-Lys] is the second most common Hb variant in humans and is the most common Hb variant in Asia. It is completely symptomless in a heterozygote form but a homozygous state is usually associated with a microcytosis similar to that observed in a β-thalassemia trait. This is because based on the molecular basis, the βE mutation is a β+-thalassemia allele [2]. It has been shown that homozygous Hb E is associated with variable Hb F level, ranging from less than 1% to approximately

Conflict of interest

There are no conflict of interests to declare.

Acknowledgments

This work was supported by grants from the National Research University (NRU) program of Khon Kaen University and the Office of the Higher Education Commission, Ministry of Education. N.P. is supported by the CHE-PhD Scholarship program of the Office of Higher Education Commission, Ministry of Education, Thailand. We are indebted to M. Benoit Sevcik from the international department of GH Henri Mondor for his help in arranging the work of N.P. in France.

References (33)

  • G. Lettre et al.

    DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

    Proc. Natl. Acad. Sci. U. S. A.

    (2008)
  • L.E. Creary et al.

    Genetic variation on chromosome 6 influences F cell levels in healthy individuals of African descent and HbF levels in sickle cell patients

    PLoS One

    (2009)
  • C.C. So et al.

    The HBS1L-MYB intergenic region on chromosome 6q23 is a quantitative trait locus controlling fetal haemoglobin level in carriers of beta-thalassaemia

    J. Med. Genet.

    (2008)
  • S. Menzel et al.

    Genetic architecture of hemoglobin F control

    Curr. Opin. Hematol.

    (2009)
  • R.A. Pandit et al.

    Association of SNP in exon 1 of HBS1L with hemoglobin F level in beta0-thalassemia/hemoglobin E

    Int. J. Hematol.

    (2008)
  • V.G. Sankaran et al.

    Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A

    Science

    (2008)
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