Chapter 1
Beginnings: the molecular pathology of hemoglobin

Douglas Higgs and Mohsin Badat

The Laboratory of Gene Regulation MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK

• Introduction
• Normal structure and function of hemoglobin and the globin genes
• Molecular pathology of the globin genes
• The pathophysiology and clinical phenotypes of thalassemia
• Conclusion
• Further reading

Introduction

The study of hemoglobin and its disorders (hemoglobinopathies) is inextricably linked to the development of molecular medicine in general. Prominent among these disorders are the thalassemias. With ~60 000 births of seriously affected individuals per year, thalassemias are among the most common group of monogenic disorders worldwide. They are caused by reduced production of the a- and ß-globin chains that form the essential tetrameric oxygen-carrying molecule hemoglobin (a2ß2). The major hemoglobinopathies, a thalassemia, ß thalassemia, and sickle cell disease, were first recognized as independent clinical entities throughout the last century. Linus Pauling first used the term "molecular disease" in 1949, after the discovery that the protein structure of sickle cell hemoglobin differed from that of normal hemoglobin. Indeed, it was this seminal observation that led to the concept of "molecular medicine," the description of disease mechanisms at the molecular level. However, until the development of recombinant DNA technology, starting in the mid-1970s, knowledge of genome structure and regulation was based on microscopy and analysis of the structure and function of proteins. However, as soon as it became possible to isolate and study human genes directly, the picture changed dramatically.

The globin genes provided the first examples of how mammalian genes are regulated. This was mainly because erythroid cells could be easily purified from the peripheral blood and the earliest erythroid cells released into the circulation (reticulocytes) contained abundant (>90%) amounts of a- and ß-globin messenger RNA. Following the discovery of RNA-dependent reverse transcriptase (by Howard Temin and David Baltimore), it became possible to radioactively label DNA transcripts derived from highly enriched globin RNA isolated from reticulocytes and use this to probe and analyze DNA and RNA from normal individuals and those with the well-defined clinical and biochemical features of the various hemoglobinopathies. Subsequently, as the ability of molecular biology to address all aspects of genetics and gene expression has advanced apace, many of the key insights into mammalian gene regulation in health and disease have been first established by studying the globin genes. These advances include the discovery of mammalian enhancers, promoters, and insulators; the discovery of splicing in mammals; the identification of termination of transcription and processing of RNA by polyadenylation. Importantly, the globin genes pioneered the idea of regulation of gene expression by long-range enhancers. The globin genes were also used to establish many of the principles by which mammalian mRNA is translated. By the late 1970s, these new discoveries paved the way for the application of molecular biology to perform a prenatal diagnosis to enable genetic counseling and prevent serious genetic diseases such as the most severe forms of thalassemia. Today, the most recent attempts to cure genetic diseases by gene therapy and gene editing are also being pioneered by the globin field.

By 2003, the first draft of the three billion bases comprising the entire human genome was announced. Together with exponential increases in the different techniques available to analyze the genome, the epigenome, the transcriptome, and the proteome, it has become possible to examine in detail virtually any gene in health and disease. Again, the hemoglobinopathies have first illustrated how co-inheritance of mutations in the transcriptional, epigenetic, and proteomic landscape can explain the different penetrance of the hemoglobinopathies in individuals with identical mutations in the globin genes. For example, we now know of variants in several non-globin genes which can change the phenotype of a severe hemoglobinopathy into a relatively mild condition. In summary, understanding how the globin genes are normally regulated and how this is perturbed in the hemoglobinopathies provides a sound basis for understanding the principles underlying molecular medicine in general.

Normal structure and function of hemoglobin and the globin genes

The structure and function of hemoglobin

The hemoglobin molecule is a tetramer consisting of two a-like and two ß-like globin chains. The varying oxygen requirements during embryonic, fetal, and adult life are reflected in the synthesis of different hemoglobin tetramers at each stage of human development (Figure 1.1A). However, they all have the same general structure, consisting of two different pairs of globin chains, each attached to one heme molecule (Figure 1.1B). Adult and fetal hemoglobins have a chains combined with ß chains (Hb A, a2ß2), d chains (Hb A2, a2d2), and ? chains (Hb F, a2?2). In embryos, a-like chains called ? chains combine with ? chains to produce Hb Portland (?2?2), or with e chains to make Hb Gower 1 (?2e2), while a and e chains form Hb Gower 2 (a2e2). Fetal hemoglobin is heterogeneous; there are two varieties of ? chain that differ only in their amino acid composition at position 136, which may be occupied by either glycine or alanine; ? chains containing glycine at this position are called G? chains, those with alanine A? chains (Figure 1.1C).

Figure 1.1 (A) Globin production at the a- and ß-globin loci during gestation and postnatal life. ?- and e-Globin are the first chains to be expressed during primitive erythropoiesis in the yolk sac, followed soon after by a- and ?-globin at approximately eight weeks' gestation. ß-globin is expressed at low levels antenatally, but switches with ?-globin at zero to six months postnatally. (B) The hemoglobin tetramer comprised of two pairs of a- and ß-globin chains, each with a prosthetic heme molecule. (C) Schematic of the a- and ß-globin chain loci showing the genes and their cognate enhancers. The genes at the a-globin locus are located downstream of four enhancers (MCS 1-4), of which MCS-2 is the most significant. MCS 1-3 lie within the introns of the gene NPRL3. The ß-globin-like genes are similarly located downstream of five regulatory elements LCR 1-5. The various hemoglobin products and their globin chain compositions are shown between the loci. (D) The oxygen dissociation curve of adult hemoglobin with modifying factors.

The synthesis of hemoglobin tetramers consisting of two unlike pairs of globin chains is absolutely essential for the effective function of hemoglobin as an oxygen carrier. The classical sigmoid shape of the oxygen dissociation curve, which reflects the allosteric properties of the hemoglobin molecule, ensures that, at high oxygen tensions in the lungs, oxygen is readily taken up and later released effectively at the lower tensions encountered in the tissues (Figure 1.1D). The shape of the curve is quite different to that of myoglobin, a molecule that consists of a single globin chain with heme attached to it, which, like abnormal hemoglobins that consist of homotetramers of like chains, has a hyperbolic oxygen dissociation curve.

The transition from a hyperbolic to a sigmoid oxygen dissociation curve, which is absolutely critical for normal oxygen delivery, reflects cooperativity between the four heme molecules and their globin subunits. When one of them takes on oxygen, the affinity of the remaining three increases markedly; this happens because hemoglobin can exist in two configurations, deoxy(T) and oxy(R), where T and R represent the tight and relaxed states, respectively. The T configuration has a lower affinity than the R for ligands such as oxygen. At some point during the addition of oxygen to the heme molecules, the transition from the T to the R configuration occurs and the oxygen affinity of the partially liganded molecule increases dramatically. These allosteric changes result from interactions between the iron of the heme groups and various bonds within the hemoglobin tetramer, which lead to subtle spatial changes as oxygen is taken on or given up.

The precise tetrameric structures of the different human hemoglobins, which reflect the primary amino acid sequences of their individual globin chains, are also vital for the various adaptive changes that are required to ensure adequate tissue oxygenation. The position of the oxygen dissociation curve can be modified in several ways. For example, oxygen affinity decreases with increasing CO2 tension (the Bohr effect). This facilitates oxygen loading to the tissues, where a drop in pH due to CO2 influx lowers oxygen affinity; the opposite effect occurs in the lungs. Oxygen affinity is also modified by the...