From
`
`by guest
`www.bloodjournal.org
`
`
`
`
`For personal use only.on January 13, 2016.
`
`ASH 50th anniversary review
`
`Hemoglobin research and the origins of molecular medicine
`Alan N. Schechter1
`
`1Molecular Medicine Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD
`
`Much of our understanding of human
`physiology, and of many aspects of pa-
`thology, has its antecedents in laboratory
`and clinical studies of hemoglobin. Over
`the last century, knowledge of the genet-
`ics, functions, and diseases of the hemo-
`globin proteins has been refined to the
`molecular level by analyses of their crys-
`tallographic structures and by cloning
`and sequencing of their genes and sur-
`Introduction
`
`rounding DNA. In the last few decades,
`research has opened up new paradigms
`for hemoglobin related to processes such
`as its role in the transport of nitric oxide
`and the complex developmental control
`of the ␣-like and ␤-like globin gene clus-
`ters. It is noteworthy that this recent work
`has had implications for understanding
`and treating the prevalent diseases of
`hemoglobin, especially the use of hy-
`
`droxyurea to elevate fetal hemoglobin in
`sickle cell disease. It is likely that current
`research will also have significant clinical
`implications, as well as lessons for other
`aspects of molecular medicine, the origin
`of which can be largely traced to this
`research tradition.
`(Blood. 2008;112:
`3927-3938)
`
`During the past 60 years, the study of human hemoglobin, probably
`more than any other molecule, has allowed the birth and maturation
`of molecular medicine. Laboratory research, using physical, chemi-
`cal, physiological, and genetic methods, has greatly contributed to,
`but also built upon, clinical research devoted to studying patients
`with a large variety of hemoglobin disorders. During this period,
`the pioneering work of Linus Pauling, Max Perutz, Vernon Ingram,
`Karl Singer, Herman Lehmann, William Castle, Ruth and Reinhold
`Benesch, Titus Huisman, Ernst Jaffe´, Ernest Beutler, and many
`others still active has been instrumental in these studies. Our
`understanding of the molecular basis of hemoglobin developmental
`and genetic control, structure-function relations, and its diseases
`and their treatment is probably unparalleled in medicine. Indeed,
`this field, especially during the first 25 years of the existence of the
`American Society of Hematology, provided the model for develop-
`ments in many other areas of research in hematology and other
`subspecialities. This review attempts to highlight some recent
`developments in hemoglobin research most relevant to the hema-
`tologist in the context of the current understanding of the functions
`of these proteins and their genes. I am occasionally asked, “What’s
`new in hemoglobin?” I believe that this review will show that we
`are still learning much that is very relevant to our understanding of
`human physiology and disease.
`
`Hemoglobin structure
`
`The human hemoglobin molecules are a set of very closely related
`proteins formed by symmetric pairing of a dimer of polypeptide
`chains, the ␣- and ␤-globins, into a tetrameric structural and
`functional unit. The ␣2␤2 molecule forms the major adult hemoglo-
`bin. Their main function in mammals is to transport oxygen (O2)
`from the lungs to tissues, but they also specifically interact with the
`3 other gases, carbon dioxide (CO2), carbon monoxide (CO), and
`nitric oxide (NO), that have important biological roles.
`The functional properties of hemoglobin molecules are primar-
`ily determined by the characteristic folds of the amino acid chains
`
`of the globin proteins, including 7 stretches of the peptide ␣-helix
`in the ␣-chains and 8 in the ␤-chains (Figure 1).1,2 These helices are
`in turn folded into a compact globule that heterodimerizes and then
`forms the tetramer structure.3 These 4 polypeptides of the hemoglo-
`bin tetramer each have a large central space into which a heme
`prosthetic group, an iron-protoporphyrin IX molecule, is bound by
`noncovalent forces, and thus the iron atom is protected from access
`of the surrounding aqueous solution. The iron atoms in this
`environment are primarily in the physiologic ferrous (FeII) chemi-
`cal valence state, coordinated to 4 pyrrole nitrogen atoms in one
`plane, to an imidazole nitrogen atom of the invariant histidine
`amino acid at position 8 of the “F”-helix, and to a gas atom on the
`side opposite (with respect to the porphyrin plane) the histidine
`residue. The reversible binding of gases to these 4 ferrous iron
`atoms in the tetramer of globin polypeptides allows hemoglobin to
`transport O2, CO, and NO.4 CO2 is transported in the blood in
`solution and by interactions with the amino-terminal residues of
`hemoglobin as a weak carbamino complex and not by binding to
`the iron atoms.
`In recent years, knowledge of the properties of the character-
`istic folds of each of the globin polypeptides and their ability to
`bind heme prosthetic groups has led to the development of a
`detailed evolutionary tree to describe the ontogeny of this
`family of genes from bacteria to vertebrates.5,6 In bacteria, they
`are known as flavohemoglobins and appear to be primarily NO
`dioxygenases for detoxifying NO; in the protist and plant taxa,
`these single-chain globin proteins are largely involved with
`electron transfer and O2 storage and scavenging. In inverte-
`brates, the O2 transport function of the globins develops as do
`several other biochemical functions. It is in the vertebrate taxa
`that the characteristic pattern of highly expressed intracellular
`globins, frequently functioning as multimers, for oxygen trans-
`port over relatively long distances evolved (Figure 2). These
`several globin proteins also include, however, the single-chain
`myoglobin, in high concentrations in many muscle tissues, as
`well as the homologous (to myoglobin and to each other) ␣- and
`
`Submitted April 22, 2008; accepted July 18, 2008. DOI 10.1182/blood-
`
`2008-04-078188.
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`3927
`
`GENENTECH 2012
`GENZYME V. GENENTECH
`IPR2016-00383
`
`

`
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`by guest For personal use only.on January 13, 2016.
`
`
`
`3928
`
`SCHECHTER
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`Figure 1. The X-ray determined structure of the
`hemoglobin molecule and a representation of its
`very high concentration in the erythrocyte. (A) The
`arrangement of the ␣-helices (shown as tubes) in each
`␣␤ unit—one on the left and one, 180° rotated, on the
`right—is shown, as are the 4 heme groups with their
`iron atoms where gas molecules bind. The site of the
`sickle mutations on mutant ␤-chains as well as the ␤93
`conserved cysteine residues is also shown. Hemoglo-
`bin molecules in the red blood cell, shown in an inset on
`the right, are very tightly packed (at a concentration of
`approximately 34 g/dL) and have little access to sol-
`vent; this allows efficient oxygen transport by each cell
`but also affects the chemical behavior of the molecules,
`such as promoting sickle cell hemoglobin polymeriza-
`tion upon slight deoxygenation. (B) A representation of
`the quaternary structural changes in the hemoglobin
`tetramer, in a top-down view, in the transition from the
`oxy conformation (left) to the deoxy conformation (right).
`The iron atoms shift relative to the planes of the heme
`groups and a central cavity between the ␤-chains
`opens, facilitating 2,3 BPG binding. These diagrams
`are based on drawings of Irving M. Geis. Illustration by
`Alice Y. Chen.
`
`␤-globins and their very stable ␣/␤ dimers that pair to form
`hemoglobin. In the highly specialized mammalian enucleated
`cell, the erythrocyte, these molecules are expressed at very high
`concentrations (Figure 1), resulting in a tremendously efficient
`
`transport mechanism. The genes of myoglobin (and other
`globins) separated from the ␣- and ␤-globin genes during
`vertebrate evolution, and these 2 genes themselves evolved into
`complex genetic loci on separate chromosomes. The numbers of
`
`Figure 2. A diagram of the proposed evolutionary
`relationships of the human globin proteins as in-
`ferred from sequence analyses. NGB, neuroglobin;
`CYGB, cytoglobin; MB, myoglobin. Reprinted from
`Pesce et al (EMBO Rep. 2002;3:1146-1151) with per-
`mission. Illustration by Alice Y. Chen.
`
`

`
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`by guest For personal use only.on January 13, 2016.
`
`
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`HEMOGLOBIN GENETICS, FUNCTIONS, AND DISEASES
`
`3929
`
`these genes, their chromosomal locations, and their developmen-
`tal control vary greatly among species; however,
`the basic
`globin gene structure and protein folds are conserved in
`evolution among all mammals.
`Myoglobin has a very high affinity for O2 compared with
`hemoglobin, but a detailed understanding of its function has still
`not been achieved. Mice with knockout of the myoglobin gene
`have almost normal physiology. Myoglobin is thought to serve
`more to facilitate oxygen diffusion in muscle, especially to
`mitochondria, than to act as a storage site, as previously thought.7
`Myoglobin also appears to act as an NO dioxygenase and a nitrite
`reductase. In the last decade, several other homologous proteins—
`neuroglobin and cytoglobin—have been detected in low amounts
`in certain tissues and appear to protect against hypoxia; again,
`however, there is much controversy about their functions.8,9
`
`Hemoglobin function
`
`The role of erythrocyte-encapsulated hemoglobin in transporting
`oxygen has been the focus of many of the greats of physiology,
`including Christian Bohr, August Krogh, J. B. Haldane, F. J. W.
`Roughton, and others in the last century and has been reviewed in
`detail.10,11 More recently elucidated was how this finely tuned
`system is regulated via heterotropic interactions with other mol-
`ecules, such as protons, anions, and bisphosphosphoglyceric acid
`in the older convention, 2,3 DPG),12 and by
`(2,3 BPG or,
`intramolecular, or homotropic, interactions for optimal normal
`respiratory function.13 Cooperative oxygen binding can be ex-
`plained very precisely in terms of the allosteric model14 of protein
`regulation of Monod, Wyman, and Changeux, but alternative
`models are still being developed.15 Understanding the physiologic
`fine-tuning of this function by proton binding (the Bohr effect) or
`2,3 BPG binding has been a triumph of basic protein chemistry and
`applied physiology during the last 50 years.10,11
`In the 1950s, the methods of protein sequence determination
`and X-ray crystallography allowed the determination of the amino
`acid sequences of various hemoglobins and the spatial arrange-
`ments of their atoms. This work, marked in particular by the
`high-resolution structural analysis—among the first for any pro-
`tein—by Nobelist Max Perutz (Figure 3) and his colleagues in the
`late 1960s,2,16 soon resulted in a detailed explanation of the
`relationship of hemoglobin function as an oxygen transporter to its
`molecular structure. Furthermore, this information allowed for the
`explanation of the clinical phenotypes of most of the many
`hundreds of characterized mutations in the globin genes and
`proteins, which cause changes in function and include the many
`“hemoglobinopathy” diseases, in terms of this molecular structure.
`These correlations, initiated by Perutz and Lehmann17 and pio-
`neered in the United States by Ranney, Beutler, Nathan, Bunn,
`Forget, and others, remain among the landmark accomplishments
`of the then-new field of molecular medicine. Although much of this
`information is now securely rooted in the textbooks, studies of
`hemoglobin function have recently become quite active again.
`In the last decade, there has been considerable attention to
`understanding the interactions of normal hemoglobin with CO, in
`recognition of the fact that as well as being a toxic hazard, CO is
`produced in the body from free heme by heme oxygenase and can
`itself activate soluble guanylyl cyclase.18 For this and other
`reasons, it has potential pharmacological applications. This atten-
`tion to non–oxygen-related functions has been even more appli-
`cable to the study of NO/hemoglobin interactions since the
`
`Figure 3. A photograph of Max F. Perutz (1914-2002) demonstrating an early
`model of the structure of hemoglobin. He devoted more than a half-century to the
`study of the detailed molecular structure of hemoglobin but was always directly
`concerned with the relevance of his work to understanding its function and its role in
`human disease. Courtesy of the Medical Research Council (London, United Kingdom).
`
`important realization in the mid-1980s that NO is a ubiquitously
`produced cell signaling molecule, acting via both soluble guanylyl
`cyclase production of cyclic GMP and other mechanisms, throughout
`almost all life forms. It is especially important in mammals in the
`regulation of vascular tone, cell interactions, and neural function.19
`It has been known since before World War I that NO reacts with
`oxyhemoglobin to produce methemoglobin, with ferric (FeIII) iron
`and nitrate ions. Recent work suggests that most of the methemoglo-
`bin circulating in red blood cells is derived from this oxidation
`process,20 which is normally reversed by the erythrocytic methemo-
`globin reductase system. In the past 40 years, a second reaction of
`NO with deoxyhemoglobin to form nitrosyl(heme)hemoglobin
`(NO-hemoglobin), with the NO liganded to the ferrous iron atom,
`has also been studied intensively. Like the reaction with oxyhemo-
`globin, this reaction had generally been assumed to be irreversible.
`However, there is now evidence that NO-hemoglobin in the circulating
`red blood cell may be capable of releasing NO molecules—thus
`potentially allowing a mechanism for hemoglobin-based, endocrine-like
`transport of NO from one tissue to another within the body.21
`Ten years ago, a third reaction of NO with oxyhemoglobin was
`postulated to be physiologically important: the binding of NO to
`the strongly conserved ␤-chain cysteine amino acid at position 93
`(Figure 1) to form S-nitrosylhemoglobin (SNO-hemoglobin).22 It
`was suggested that SNO-hemoglobin can physiologically dissoci-
`ate to release NO at low oxygen concentrations. Thus, this could be
`a mechanism for homeostatic control of blood flow to tissues,
`because the NO released would promote vascular dilatation and
`increase blood flow and oxygen delivery. This hypothesis, although
`teleologically attractive, has been very controversial, with many
`studies negating it, and very recent work with transgenic mice
`lacking the ␤93 cysteine residues appears to disprove it.23 More
`recently, an alternate hypothesis to account for the transport of NO
`by erythrocytes has been advanced. It has been suggested that
`
`

`
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`by guest For personal use only.on January 13, 2016.
`
`
`
`3930
`
`SCHECHTER
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`Figure 4. A representation of nitric oxide (NO)/hemoglobin reactions in the arterial microcirculation. Reactions that appear to predominate under physiologic conditions
`(center), as well as pathologic lesions due to hemolysis (right) and results of high or pharmacologic levels of NO (left) are indicated. Under basal conditions, NO (a short-lived
`free radical) produced by endothelial NO synthase enzymes largely diffuses into surrounding smooth muscle to activate soluble guanylyl cyclase (sGC) to produce cyclic GMP
`and regulate vascular tone. The interactions of NO with red cells under these conditions seem to be limited by several barriers to diffusion, at the red cell membrane and
`streaming of plasma near the endothelium. With hemolysis (or with administration of hemoglobin-based blood substitutes), cell-free oxyhemoglobin acts as an efficient
`scavenger of NO, causing vasoconstriction and perhaps pathological organ conditions. When endogenous NO levels become very high, or when it is administered by inhalation
`or by infusion of nitrite ions or other NO donors, reactions in the plasma and within erythrocytes become very important. Reactions with oxygen will tend to oxidize NO to nitrite
`and nitrate. Reactions with plasma molecules will form thiol (SNO) compounds and other species; plasma nitrite can also be reduced by endothelial xanthine oxidoreductase
`(XOR) to NO. Small amounts of SNO-Hb form, but its function is not at all clear. Nitrite from the plasma may enter the red cell or be formed in the cell itself, where reactions with
`hemoglobin and the ascorbate cycle can reduce it to NO. Although the prevalent reactions with oxyhemoglobin to form methemoglobin and nitrate tend to destroy NO
`bioactivity,
`these other reactions may allow its preservation and modulation for physiologic functions. Adapted from Schechter and Gladwin (N Engl J Med.
`2003;348:1483-1485), with permission. Illustration by Alice Y. Chen.
`
`nitrite ions within erythrocytes can be reduced to NO by
`deoxyhemoglobin—with reaction kinetics maximal at approximately
`50% oxygen saturation—so that NO is increasingly generated as red
`blood cells enter regions of relative hypoxia.24 Thus, there are now
`several potential explanations for a likely central function of NO in
`controlling blood flow via hypoxic vasodilation (Figure 4).
`These recent studies of NO interactions with hemoglobin point
`to the increasing realization in the last few years that hemoglobin
`has evolved with functional properties important for the physiology
`of several gases, especially NO, as well as that of the paradigmatic
`delivery of O2. There is also some indication that abnormalities in
`hemoglobin levels or localization (for example, the increases in
`total intracellular hemoglobin that occur in polycythemia or of
`cell-free hemoglobin in chronic and acute anemias [Figure 4]) may
`result in clinical abnormalities because of their overall tendency to
`deplete available NO. The major toxicities of all hemoglobin-based
`blood substitutes seem to be similar and are likely to be due largely
`to enhanced destruction of NO by the cell-free hemoglobin25 but
`could possibly be overcome by replacement of the NO.26
`
`The hemoglobin phenotype
`
`In erythrocytes of normal human adults, hemoglobin A (␣2␤2)
`accounts for approximately 97% of the protein molecules, hemoglo-
`bin A2 (␣2␦2) for 2%, and hemoglobin F or fetal hemoglobin (␣2␥2)
`for 1% (Figure 5). This distribution reflects the patterns of
`expression of the ␣-globin gene locus on human chromosome 16
`and the ␤-globin gene locus on human chromosome 11. After the
`evolutionary separation of the 2 mammalian globin loci, each locus
`
`has undergone complex changes that resulted in the presence of
`multiple genes and nonexpressed pseudogenes in the human
`genome. The pattern of expression of these genes shifts from the
`more 5⬘ genes on the DNA to more 3⬘ genes during fetal, then
`neonatal, and then adult development stages (Figure 6).27 In the
`fetus, the ␨ and ε genes are initially expressed primarily in the yolk
`sac, para-aortic region, and then the liver, resulting in the formation
`of hemoglobins Gower 1, Gower 2, and Portland. Their down-
`regulation in early embryonic life is followed by the expression of
`the 2 ␣-genes and the 2 ␥-genes (G␥ and A␥); they are functionally
`identical but are different in that there is either a glycine or an
`alanine at position 136. This causes the accumulation of hemoglo-
`bin F, which predominates in the last 2 trimesters of gestation and
`has a slightly higher oxygen affinity than the adult hemoglobins
`because it binds 2,3 BPG less strongly. At birth, although the ␣
`genes remain fully active, the ␥ genes are effectively down-
`regulated and the ␤-like (␦ and ␤) genes are up-regulated so that,
`normally, by the end of the first year of life, the “adult” hemoglobin
`phenotype, hemoglobins A and A2, is predominant. In some cases,
`expression of the ␥-globin persists in adult erythroid cells; this
`largely asymptomatic state is known as hereditary persistence of
`fetal hemoglobin (HPFH).28
`The covalent modification of the major adult hemoglobin by
`nonenzymatic glycation of the ␤-chain amino-terminal residue by
`glucose forms hemoglobin A1c.29 This was observed in electro-
`phoretic studies of hemoglobin phenotypes and has opened a vast
`area of diabetes-related research. There has also been much
`progress in understanding the diverse causes, manifestations, and
`treatment of methemoglobinemia.30,31 Indeed, the discovery of a
`deficiency of cytochrome b5 reductase (methemoglobin reductase)
`
`

`
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`by guest For personal use only.on January 13, 2016.
`
`
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`HEMOGLOBIN GENETICS, FUNCTIONS, AND DISEASES
`
`3931
`
`Figure 5. The genomic structure of the clusters of
`␣-like and ␤-like globin genes, on chromosomes 16
`and 11, in human beings. The functional ␣-like genes
`are shown in dark blue and the pseudogenes are in
`light blue; 2 of these (␮ and ␪-1) code for small amounts
`of RNA. The functional ␤-like genes are shown in light
`green. The important control elements, HS-40 and the
`LCR, discussed in the text, are also shown at their
`approximate locations. The ␣-gene cluster is approxi-
`mately two thirds of the length of the ␤-gene cluster; it is
`transcribed from telomere toward centromere, the oppo-
`site of the ␤ cluster. The various hemoglobin species
`that are formed from these genes, with their prime
`developmental stages, are shown in the lower part of
`the figure. Illustration by Alice Y. Chen.
`
`as a cause of familial methemoglobinemia may be considered the
`first description of an enzyme defect in a hereditary disorder.32
`There have also been significant advances in understanding the
`complex physiologic adaptive responses to acute and chronic
`hypoxia, especially of populations at high altitudes.33
`During the last 30 years, an enormous amount of effort has been
`devoted to understanding the molecular and cellular mechanisms
`that underlie these changes (called hemoglobin “switching”) in
`expression of the ␣- and ␤-globin gene clusters.34 This has been
`because of the intrinsic interest of this system as one of developmen-
`tal gene control but also because of the potential relevance of this
`information to developing therapies for the 2 most common groups
`of genetic diseases of hemoglobin, the sickle cell syndromes and
`the thalassemia syndromes. Before reviewing these studies of
`globin developmental control, I note some of the relevant work—
`especially recent findings—on the pathophysiology of these 2 groups
`of diseases and how altering the hemoglobin phenotype might be
`clinically beneficial.
`
`Sickle cell disease
`
`The discovery by Linus Pauling and his associates in 194935 that
`the molecular basis of sickle cell anemia is due to an abnormal
`
`hemoglobin virtually created the field of molecular medicine
`and moved research hematology to its forefront. It is sometimes
`forgotten that this molecular medicine paradigm also required
`understanding of the inheritance pattern of this disease, which
`was supplied in the same year by J. V. Neel,36 whose publication
`is also one of the founding articles of the field of medical
`genetics. We now have a detailed understanding of how a single
`nucleotide change (A to T) in the ␤-globin gene leads to the
`valine for glutamic acid substitution37 in the ␤-globin protein.
`This in turn allows the formation of stable intermolecular
`interactions (linear polymers of the tetramers) in the concen-
`trated intracellular solutions of deoxyhemoglobin S (␣2␤2
`S or
`sickle hemoglobin).38 This process is the basis for our understand-
`ing of the pathophysiology of this disease39,40 at the genetic,
`molecular and cellular levels. Sickle cell anemia pathophysiol-
`ogy is a consequence of
`this reduced solubility, causing
`polymerization of hemoglobin S tetramers in red blood cells
`upon partial deoxygenation and the impaired flow of these cells
`in the microcirculation.38 Other mechanisms secondary to
`intracellular polymerization have been extensively studied,
`especially in animal models, but their relative importance to
`human pathophysiology remains unclear.
`
`Figure 6. The timeline of the expression of the human globin genes
`from early stages of fetal development to the changes that occur at
`birth and in the first year of life. Also shown are the major sites of
`erythropoiesis and the types of hemoglobin-containing cells during these
`periods. These analyses are largely based on observations of clinical
`samples made by Huehns et al in the 1960s; the figure is reprinted from
`Wood (Br Med Bull. 1976;32:282) with permission. Illustration by Alice Y.
`Chen.
`
`

`
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`by guest For personal use only.on January 13, 2016.
`
`
`
`3932
`
`SCHECHTER
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`Figure 7. A diagram of the postulated effect of
`hydroxyurea in inhibiting hemoglobin S-polymeriza-
`tion, by increasing hemoglobin F levels (shown as
`25%) in each sickle erythrocyte and thus decreas-
`ing the degree of microvascular obstruction at any
`oxygen level. The sparing effect of hemoglobin F,
`greater than that of hemoglobin A, occurs because the
`mixed hybrid ␣2␤S␥ that forms inside the red cell does
`not enter the polymer phase. Adapted from Schechter
`and Rodgers (N Engl J Med. 1995; 334:333-335), with
`permission. Illustration by Alice Y. Chen.
`
`More than 50 years ago it was postulated (probably first by
`J. B. S. Haldane) that the sickle mutation results in increased
`resistance to malaria in heterozygotic persons or carriers41; subse-
`quent work indicates that this is true for thalassemia as well.42
`Current research suggests the importance of redox and immuno-
`logic processes in this protection, but the exact cellular mecha-
`nisms are not yet clear.42,43 Again, as with so many other studies of
`the clinical biology of hemoglobin,
`this concept of selective
`advantages for carriers of certain disease-causing (in the homozy-
`gous state) genes has been applied widely.
`Another new concept from sickle cell anemia research quickly
`extended to other diseases was the realization by Y. W. Kan and his
`colleagues in 197844 that restriction enzymes could be used to
`detect DNA polymorphisms linked to the abnormal ␤-globin gene
`to identify prenatally those fetuses who have one or both of the
`mutant hemoglobin genes. These studies also initiated the gradual
`transition of the molecular diagnosis of hemoglobin disorders from
`protein methods to the current wide range of extremely sensitive
`and precise nucleic acid analyses.45
`However, despite the detailed characterization of the abnormal
`gene and protein and the behavior of hemoglobin S in red cells, we
`understand relatively little of how these abnormalities affect
`specific organs and the overall health of affected persons. The best
`indicator of this conundrum is the unexplained heterogeneity in age
`of onset and severity of disease in persons whose hemoglobin
`genotype and phenotype appear similar or identical.46 Unlike
`“classical” monozygotic diseases, even many of the thalassemic
`syndromes, clinical progression and the need for treatment in
`patients with sickle cell anemia patients can only be predicted in
`limited circumstances, such as in children detected to have
`abnormal blood flow in the large vessels of the brain as measured
`by the transcranial-Doppler ultrasound method47 or in adults with
`pulmonary hypertension.48
`Although many other measurements, such as globin cluster
`haplotype analysis or white blood cell levels, have been suggested
`to have explanatory and predictive value, only 2, the presence of
`␣-thalassemia and the levels of hemoglobin F, have been validated
`comprehensively. Coexisting ␣-thalassemia leads to a reduction in
`MCHC, which inhibits hemoglobin S polymerization, but this beneficial
`
`effect seems to be counter-balanced by the increase in total hemoglobin
`levels, which may have some deleterious effects.49,50
`The beneficial effects of hemoglobin F have been confirmed by
`clinical observations, epidemiologic studies, biophysical measure-
`ments, and therapeutic trials. In 1948, Janet Watson51 noted that
`until adult hemoglobin displaces the form present at birth (hemoglo-
`bin F), manifestations of sickle cell disease are limited. Population
`studies among different groups of persons with sickle cell disease
`(eg, Saudi Arabs vs African populations) or within single geo-
`graphic areas, as well as a large “natural history” study in the
`United States confirmed that various measures of severity were
`inversely related to hemoglobin F levels but suggested that very
`high (⬎ 25%) levels were needed for major benefit. At the same
`time, diverse laboratory studies showed the mechanism by which
`hemoglobin F had a sparing effect on intracellular polymerization
`(Figure 7) and confirmed clinical estimates of the levels of
`hemoglobin F needed for benefit.52 Equally importantly, several
`drugs were found to increase hemoglobin F levels in nonhuman
`primates. DeSimone and Heller53 and Letvin et al54 showed that
`5-azacytidine and hydroxyurea (now frequently designated as
`hydroxycarbamide) had such effects. This work was extended to
`patients by Platt, Charache, Dover, Nienhuis, Ley, Rodgers, and
`their colleagues (reviewed by Rodgers55). In a multicenter, double-
`blinded study of adults with frequent pain crises, led by Charache,56
`hydroxyurea improved several clinical parameters compared with
`placebo. In 1998, hydroxyurea was approved by the US Food and
`Drug Administration for treating these types of patients, and a
`recent systematic review has confirmed its efficacy in adult patients
`with sickle cell disease.57
`However, many patients do not respond at all to hydroxyurea
`with elevations of hemoglobin F, whereas some clinical manifes-
`tations seem to be little affected by even the 10% to 15% levels
`of hemoglobin F obtained in some patients with the drug.
`Furthermore, there is yet limited evidence that the drug prevents
`damage of crucial organs, such as the lungs, kidneys, and brain,
`or improves survival in adult patients.58 Long-term and con-
`trolled studies in children to assess the effects and safety of
`hydroxyurea have only been recently initiated. Thus, in addition
`to further clinical outcome studies with hydroxyurea, there is a
`
`

`
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`by guest For personal use only.on January 13, 2016.
`
`
`
`BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
`
`HEMOGLOBIN GENETICS, FUNCTIONS, AND DISEASES
`
`3933
`
`strong need to find other agents that may singly or in combina-
`tion with hydroxyurea have a more robust effect on hemoglobin
`F levels. To this end, studies with erythropoietin, butyrate
`compounds, and deoxyazacytidine are being pursued, but at
`present, none seems strongly promising. Clearly, much more
`work, both clinical and laboratory, is needed to test these drugs
`further and to find new agents if we are to improve on this
`partially effective pharmacologic approach to this disease.
`Although the utility of hydroxyurea in treating sickle cell
`disease has been generally accepted in the academic community,
`its more general use in the treatment of patients with sickle cell
`disease has been quite limited, even among patients who are
`likely to benefit.59 Further complicating its evaluation has been
`that
`its clinical effects have been attributed by some to
`mechanisms other than inducing hemoglobin F, such as lowering
`neutrophil counts or adhesion molecules, generating NO, and
`others. The evidence for these is minimal, and some of these
`effects may be indirect results of elevating hemoglobin F.
`Likewise, many pathophysiologic mechanisms have been pro-
`posed in sickle cell disease studies as alternatives or comple-
`ments to the intracellular polymerization of deoxyhemoglobin S
`and its effects on the rheologic properties of
`the sickle
`erythrocyte. None has the weight of evidence that surrounds the
`primary polymerization phenomenon, and they are likely to be
`secondary factors; none of the therapeutic approaches based on
`these hypotheses has been promising up to now compared with
`inhibiting polymerization of deoxyhemoglobin S with hemoglobin F.
`However, it has recently been proposed that as a consequence
`of the fragility of the sickle erythrocyte (due to intracellular
`polymerization, which results in intravascular hemolysis and the
`chronic anemia characteristic of this disease), circulating cell-
`free hemoglobin levels are increased, and this acts as a strong
`NO scavenger. This hypothesis60 postulates that some of the
`clinical manifestations of sickle cell disease (pulmonary hyper-
`tension,
`leg ulcers, and possibly stroke) relate to this NO
`deficiency, whereas others (vaso-occlusive pain crises, acute
`chest syndrome) are due primarily to occlusion of microcircula-
`tory flow by red cells made rigid (not necessarily “sickled”) by
`intracellular polymer. Patients with sickle cell disease appear to
`differ in the relative importance of the hemolytic and the
`occlusive mechan