Insight of fetal to adult hemoglobin switch: Genetic modulators and therapeutic targets

Abstract

The clinical heterogeneity of β-hemoglobinopathies is so variable that it prompted the researchers to identify the genetic modulators of these diseases. Though the primary modulator is the type of β-globin mutation which affects the degree of β-globin chain synthesis, the co-inheritance of α-thalassemia and the fetal hemoglobin (HbF) levels also act as potent secondary genetic modifiers. As elevated HbF levels ameliorate the severity of hemoglobinopathies, in this review, the genetic modulators lying within and outside the β-globin gene cluster with their plausible role in governing the HbF levels have been summarised, which in future may act as potential therapeutic targets.

Introduction

Way back in 1866, Korber et al., discovered that the hemoglobin obtained from umbilical cord was far more resistant to alkali denaturation than the normal adult hemoglobin (HbA, α₂β₂) (Chromosome location: α globin gene-16p13.3; β globin gene-11p15.4) [1]. After almost 60 years of this discovery, in 1934 Brinkman et al., established that the variation in the alkali resistant characteristic of hemoglobin was due to the globin portion of the molecule and thus the alkali resistant hemoglobin was termed as fetal hemoglobin (HbF, α₂γ₂) (Chromosome location: γ globin gene-11p15.4) since it formed the major hemoglobin fraction of the growing fetus [2]. During the same time period, Drescher and Kiinzer, & Huehns et al., identified that the embryonic hemoglobin in small human embryos (gestational age: 7 weeks-12 weeks) had an alkali denaturation between HbA and HbF [3]. These series of discoveries left behind a perplexing question; does the hemoglobin in humans during the ontogeny undergo successive switches? And if yes, is there a perspective for molecular cure by reactivating fetal globin genes in hemoglobinopathy patients?

The possibility that hemoglobinopathies can be cured by induction of fetal hemoglobin was realized as the symptoms are seen in 6 months of age after birth when there is a gradual reduction in the HbF levels. In β-thalassemia, the main cause of the disease is an imbalance in the globin chain ratio, wherein the excess unbound α globin chain precipitates, inhibiting the maturation of the erythroid precursors, ultimately leading to ineffective erythropoiesis and anemia [4]. Thus, the continued γ-globin gene expression in β-thalassemics, may lead to a reduction in the globin chain imbalance as γ-globin synthesized will combine with the excess unbound α-globin chain [5]. Similarly, the appreciating role of fetal hemoglobin in sickle cell disease started more than 60 years ago when Janet Watson confirmed that infants with sickle cell disease with high HbF level had few symptoms and that their deoxygenated erythrocytes took a longer time to sickle and did not deform as extensively as did their sickle cell trait-carrying mother's cells [6]. The pathophysiology of sickle cell disease is dependent on the polymerization of deoxy sickle hemoglobin under low oxygen condition due to altered biophysical property, which is retarded with increased HbF concentration. Both HbF and its mixed hybrid tetramer (α₂β^Sγ) cannot enter the deoxy sickle hemoglobin polymer phase, thus circumventing the cellular damage evoked by HbS polymers [7]. In both the hemoglobinopathy conditions, increased γ-globin gene expression acts as a well-known disease modifier. Hence understanding the molecular mechanism of hemoglobin switching and identification of molecular targets, for reversing this switch, is a subject of intense research [4].

During the ontogeny, globin genes (ε, ^Gγ, ^Aγ, δ, β) in the β-globin cluster are sequentially expressed, leading to the production of different hemoglobin molecules with distinct physiological properties. The site for primary hemoglobin synthesis is the embryonic yolk sac and the first wave of hemoglobin switch (primitive to definitive) occurs in the fetal liver after 5 weeks of gestation, wherein there is a switch from embryonic (ζ₂ε₂) to fetal hemoglobin (α₂γ₂) [8]. Towards the end of 3rd trimester, there is a gradual change from fetal (α₂γ₂) to adult hemoglobin (α₂β₂). This second major hemoglobin switch occurs in the bone marrow and lasts until 6 months of age after birth [8]. Fig. 1 shows a schematic representation of different hemoglobin progressively expressed during distinct stages of development. With the dawn of molecular era, various models for gene switching have been described. These include (i) Gene competition: In this model, the γ and β globin genes during the ontogeny compete for interaction with the LCR in stage specific manner for their expression. (ii) Chromosomal looping: Here, the hypersensitive sites in LCR of the β globin cluster, loops out to come in close proximity with the appropriate gene within the cluster, for promoting its transcription. (iii) Autonomous gene silencing: It is a self-governed process where all the elements for gene silencing are located within or in near proximity to the target gene that has to be repressed. (iv) Polymerase tracking model: It is based on the idea that the enhancer-bound protein complex consisting of RNA polymerase II scrutinizes the DNA sequence until a promoter region is confronted to activate gene expression. It is hypothesized that these models collectively bring about the process of globin gene switching [8,9]. Further, the hemoglobin switching process involves a complex interaction between the cis-acting elements (locus control region of the beta-globin gene cluster) and trans-acting transcription factors which co-ordinately carry out chromatin remodelling activities [9].

The β-globin gene cluster contains a 5′ distal regulatory element known as Locus Control Region (LCR), which has 5 active DNAase I hypersensitive sites (HS) 1–5, that is required for appropriate globin gene expression. This is followed with the series of globin genes (ε, ^Gγ, ^Aγ, δ, β) arranged sequentially in their order of expression (Fig. 2). To the downstream of the β globin gene is another HS site 3’ HS1. The β-globin cluster is flanked with olfactory receptors at both 5′ and 3′ ends [10]. Wijgerde et al., proposed a competitive model for globin gene regulation, wherein the LCR participates in the long-range looping interaction, which leads to only one productive LCR-globin gene interaction [10]. The potent transcriptional activation of the LCR is also due to the clustering of various erythroid transcription factors that bind to the hypersensitivity sites which act in concert to facilitate long-range looping activity. The cis-acting promoter sequences in the individual globin genes and the LCR serve as a template for the binding of various transcription factors [10]. It is observed that naturally occurring mutations in γ globin promoter region is associated with the hereditary persistence of fetal hemoglobin phenotype (HPFH). The mechanisms underlying the continued expression of HbF with these mutations are thought to involve alterations in the protein binding motifs that ultimately ablates the binding of repressors or enhancers affecting the gene expression [11]. The most extensively studied variation in several population groups is the XmnI polymorphism (C → T) residing at −158 position (HBG2 c.-211C → T) in the ^Gγ globin promoter region. The T allele of XmnI polymorphism is linked to the raised HbF levels and milder hemoglobinopathy conditions [12]. Though T allele in its homozygous state seems to have little effect on HbF levels in normal individuals, it has been associated with an increase of HbF especially during the erythropoietic stress, suggesting its role in HbF induction [13]. In clinically milder hemoglobinopathy patients where no mutation is detected in the γ-globin promoter region, the other genetic modulators [HBS1L-MYB intergenic region, BCL11A, KLF1] leading to raised HbF phenotype could play some role [7,8].