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Classification of the Disorders of

Bernard G. Forget1 and H. Franklin Bunn2

1Section of Hematology, Department of Medicine, Yale School of Medicine, New Haven, Connecticut 06520-8028 2Hematology Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 Correspondence: [email protected]

Over the years, study of the disorders of hemoglobin has served as a paradigm for gaining insights into the cellular and molecular biology, as well as the pathophysiology, of inherited genetic disorders. To date, more than 1000 disorders of hemoglobin synthesis and/or struc- ture have been identified and characterized. Study of these disorders has established the principle of how a mutant genotype can alter the function of the encoded , which in turn can lead to a distinct clinical phenotype. Genotype/phenotype correlations have provided important understanding of pathophysiological mechanisms of disease. Before presenting a brief overview of these disorders, we provide a summary of the struc- ture and function of hemoglobin, along with the mechanism of assembly of its subunits, as background for the rationale and basis of the different categories of disorders in the classification.

n impressive degree of molecular “engineer- tion to Fe3þ, which is incapable of binding ox- Aing” was necessary for the evolution of a ygen.3 Delicate noncovalent interactions be- multisubunit protein that higher organisms re- tween unlike subunits are required for

www.perspectivesinmedicine.org quire for optimal homeostasis and buf- the hemoglobin tetramer a2b2 to bind and un- fering of acidic metabolic waste products. Each load oxygen in a cooperative manner, thereby globin subunit must form a stable linkage with assuring maximal transport to actively metab- (ferroprotoporphyrin IX) situated on the olizing cells. This phenomenon is reflected by external surface of the protein so that oxygen a sigmoid oxygen-binding curve that depends in the RBC cytosol can bind reversibly to the on hemoglobin tetramer having two quaternary ’ atoms. Moreover, the hydrophobic structures: the T or deoxy conformer that has cleft into which the heme is inserted must be low oxygen affinity and the R or oxy conformer able to protect the Fe2þ heme iron from oxida- that has high oxygen affinity. Further fine-

3For more detailed information, see Dailey and Meissner (2013). Editors: David Weatherall, Alan N. Schechter, and David G. Nathan Additional Perspectives on Hemoglobin and Its Diseases available at www.perspectivesinmedicine.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011684 Cite this article as Cold Spring Harb Perspect Med 2013;3:a011684

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B.G. Forget and H.F. Bunn

tuning of hemoglobin function comes from its of globin genes that impair synthesis allosteric behavior triggered by the binding of give rise to and anemia of varying two small effector molecules 2,3-BPG, and pro- degree. In addition, well-defined clinical and tons to specific sites on the T structure distant hematologic phenotypes are associated with from the heme groups.4 To endow the mutations that alter the structure of globin sub- with high oxygen carrying capacity hemoglo- units, discussed in more detail in Thom et al. bin must be stuffed into flexible circulating (2013). Impairment of hemoglobin solubility RBCs. A remarkably high degree of solubility can be caused either by the formation of intra- is required for hemoglobin to achieve an intra- cellular polymers () or by the cellular concentration of 34 g/dl or 5 mM development of amorphous precipitates (con- (tetramer). genital Heinz body hemolytic anemia). Abnor- To reach such a high corpuscular hemoglo- malities of oxygen binding can lead either to bin concentration it is essential that a-globin as erythrocytosis (high O2 affinity mutants) or to well as b-globin (or g-globin) mRNA be ex- cyanosis (low O2 affinity mutants). Some glo- pressed at very high levels during erythroid dif- bin mutants have structural alterations within ferentiation. Moreover, a- and non-a-globin the heme pocket that result in oxidation of synthesis must be closely matched. As explained the heme iron and pseudocyanosis because of in Nienhuis and Nathan (2012), subunit imbal- . anceis centraltothe pathophysiologyof thethal- assemias. Freea-globin subunits areparticularly toxic to erythroid cells. This threat is alleviated GENERAL CLASSIFICATION OF by the presence of a-hemoglobin stabilizing HEMOGLOBIN DISORDERS protein (AHSP), a chaperone that is expressed Hemoglobin disorders can be broadly classified at high levels in erythroid cells and binds specif- into two general categories (as listed in Table 1): ically and tightly to heme-intact a-globin sub- units (Kihm et al. 2002; Feng et al. 2004; Mollan 1. Those in which there is a quantitative defect et al. 2010, 2012). The AHSP protects the cell in the production of one of the globin sub- from potentially toxic oxidized (Fe3þ) heme un- units, either total absence or marked reduc- til it is reduced to functional Fe2þ heme by cy- tion. These are called the thalassemia syn- tochrome b5 reductase. On encountering a free dromes. heme-intact b-globin subunit, the a-globin 2. Those in which there is a structural defect in dissociates from AHSP to form the extremely one of the globin subunits. stable ab dimer. As discussed later in this www.perspectivesinmedicine.org work, this process is facilitated by electrostatic The majority of human hemoglobin mu- attraction between positively charged a-globin tants were discovered as an incidental finding, subunits and negatively charged b-globin sub- unassociated with any hematologic or clinical units. phenotype. However, a numberof a- and b-glo- In view of the multiple molecular con- bin mutants are associated with distinct clinical straints that are required for high-level produc- phenotypes. These fall into five broad catego- tion of fully functional and highly soluble he- ries: the sickle syndromes (SS, SC, Sb0-thalasse- moglobin, it is no surprise that Murphy’s law mia, and Sbþ-thalassemia); unstable mutants is in full force: whatever can go wrong will. causing congenital Heinz body hemolytic ane- As discussed briefly here, and in much more mia; mutants with high oxygen affinity result- detail in Thein (2013), Higgs (2013), Nienhuis ing in erythrocytosis; low oxygen affinity mu- and Nathan (2012), and Musallam et al. (2012), tants and the M causing cyanosis; and mutants associated with a thalassemia phe- notype. Most of these disorders are inherited 4For more detailed information on hemoglobin function, genetic defects, but there are some defects that see Schechter (2013). are acquired or occur de novo.

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Classification of Hemoglobin Disorders

Table 1. Classification of hemoglobin disorders I. QUANTITATIVE DISORDERS OF GLOBIN II. QUALITATIVE DISORDERS OF GLOBIN CHAIN SYNTHESIS/ACCUMULATION STRUCTURE: STRUCTURALVARIANTSOF The thalassemia syndromes . b-Thalassemia A. Sickle cell disorders Clinical classification: SA, sickle cell trait b-Thalassemia minor or trait SS, sickle cell anemia/disease b-Thalassemia major SC, HbSC disease b-Thalassemia intermedia S/b thal, sickle b-thalassemia disease Biochemical/genetic classification: S with other Hb variants: D, O-Arab, other b0-Thalassemia SF, Hb S/HPFH þ b -Thalassemia B. Hemoglobins with decreased stability d-Thalassemia (unstable ) g-Thalassemia Mutants causing congenital Heinz body hemolytic Lepore fusion gene anemia db-Thalassemia Acquired instability—oxidant hemolysis: Drug- 1gdb-Thalassemia induced, G6PD deficiency HPFH C. Hemoglobins with altered oxygen affinity “Dominant” b-thalassemia (structural variants with High/increased oxygen affinity states: b-thalassemia phenotype) Fetal red cells b-Thalassemia with other variants: Decreased RBC 2,3-BPG HbS/b-thalassemia Carboxyhemoglobinemia, HbCO HbE/b-thalassemia Structural variants Other Low/decreased oxygen affinity states: B. a-Thalassemia Increased RBC 2,3-BPG Deletions of a-globin genes: Structural variants One gene: aþ-thalassemia Two genes in cis: a0-thalassemia D. Methemoglobinemia Two genes in trans: homozygous aþ-thalassemia Congenital methemoglobinemia: (phenotype of a0-thalassemia) Structural variants Three genes: HbH disease b5 reductase deficiency Four genes: Hydrops fetalis with Hb Bart’s Acquired (toxic) methemoglobinemia Nondeletion mutants: E. Posttranslational modifications Hb Constant Spring Nonenzymatic glycosylation

www.perspectivesinmedicine.org Other Amino-terminal acetylation C. De novo and acquired a-thalassemia Amino-terminal carbamylation a-Thalassemia with mental retardation syndrome (ATR): Deamidation Due to large deletions on involving the a-globin genes Due to mutations of the ATRX transcription factor gene on chromosome X a-Thalassemia associated with myelodysplastic syndromes (ATMDS): Due to mutations of the ATRX gene

THE THALASSEMIA SYNDROMES subunits of hemoglobin. In the alpha (a)-thal- assemias, there is absent or decreased pro- The thalassemia syndromes are inherited dis- duction of a-globin subunits, whereas in the orders characterized by absence or markedly beta (b)-, there is absent or re- decreased accumulation of one of the globin duced production of b-globin subunits. Rare

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B.G. Forget and H.F. Bunn

thalassemias affecting the production of delta (b-thalassemia major), there is usually a severe (d)- or gamma (g)-globin subunits have also transfusion-dependent hemolytic anemia asso- been described but are not clinically signif- ciated with marked ineffective erythropoiesis icant disorders. Combined deficiency of d þ resulting in destruction of erythroid precursor b-globin subunits, or of all of the b-like glo- cells in the bone marrow. bin subunits also occurs. These disorders are A less common clinical phenotype is re- described in detail in Thein (2013) and Higgs ferred to as b-thalassemia intermedia. In this (2013). clinical disorder, there is a moderate to severe, partially compensated, hemolytic anemia that does not require chronic transfusion therapy THE b-THALASSEMIAS to maintain a satisfactory circulating hemo- globin level in the affected patient, although The b-thalassemias can be subclassified into occasional transfusions may be required if the those in which there is total absence of normal anemia worsens because of associated com- b-globin subunit synthesis or accumulation, plications. Such patients have a milder disease the b0-thalassemias, and those in which some because there is less severe a- to non-a-globin structurally normal b-globin subunits are syn- subunit imbalance than in typical b-thalasse- thesized, but in markedly decreased amounts, mia major patients, resulting in less accumu- the bþ-thalassemias. The molecular basis of the lation of free a-subunits that cause the ineffec- b-thalassemias is very heterogeneous (see Thein tive erythropoiesis. There are different possible 2013), with over 200 different mutations having causes for such a lessened a- to non-a-globin been described. In general, the mutations caus- subunit imbalance, including: inheritance of ing b-thalassemia are point mutations affect- milder bþ-thalassemia mutations with less se- ing a single nucleotide, or a small number of vere than usual deficiency of b-globin subunit nucleotides, in the b-globin gene. Rare deletion production; coinheritance of a form of a-thal- forms of b-thalassemia have also been de- assemia; or coinheritance of another genetic scribed. One of these deletions is caused by “un- trait associated with increased production of equal” crossing over between the linked and the g-subunit of . Most pa- partially homologous d- and b-globin genes, tients with b-thalassemia intermedia carry resulting in the formation of a fusion db-globin two mutant b-globin genes: they have a geno- gene, the Lepore gene, that has a low level of type typical of b-thalasemia major, but the phe- expression. Large deletions involving part or notype is modified by one of the factors listed all of the b-globin gene cluster are responsible above. However, rare cases of b-thalassemia in- www.perspectivesinmedicine.org for the db-thalassemias, the 1gdb-thalassemias, termedia are caused by heterozygosity for a and the hereditary persistence of fetal hemoglo- single mutant b-globin gene associated with bin (HPFH) syndromes. the production of a highly unstable b-globin Despite the marked heterogeneity in the subunit that causes RBC damage in a fashion molecular basis of the b-thalassemias, the clin- similar to excess free a-subunits; this is the ical phenotype of these disorders is relatively so-called “dominant b-thalassemia” (Thom et homogeneous because of their common patho- al. 2013). physiology: deficiency of HbA tetramers and The db-thalassemias are associated with to- excess accumulation of free a-subunits incapa- tal deficiency of b-globin subunit production, ble of forming hemoglobin tetramers because of but are clinically milder than typical cases of b0- deficiency of b-like globin subunits (see Nien- thalassemia, because there is an associated per- huis and Nathan 2012). In heterozygotes (b- sistent high level of expression of the g-subunit thalassemia trait or b-thalassemia minor), there of fetal hemoglobin that decreases the degree is mild to moderate hypochromic microcytic of a-subunit excess. The 1gdb-thalassemias are anemia, without evidence of hemolysis, whereas associated with neonatal hemolytic anemia, in homozygotes or compound heterozygotes but this resolves during the first few months

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Classification of Hemoglobin Disorders

of life and the associated phenotype in adults is treatment by RBC transfusion. The basis of that of b-thalassemia trait or b-thalassemia mi- the hemolysis is the excess accumulation of b- nor. The syndrome of hereditary persistence of globin subunits that self-associate to form fetal hemoglobin (HPFH) is not, strictly speak- b-chain tetramers or HbH. In contrast to the ing, a form of b-thalassemia because it is not situation in the b-thalssemias in which excess associated with significant a- to non-a-globin a-globin subunits rapidly form insoluble ag- subunit imbalance, but is characterized by high gregates, excess b-globin chains can form sol- levels of persistent g-globin production and is uble tetramers (HbH). However, HbH is rela- frequently considered within the spectrum of tively unstable and does precipitate as RBCs age, db-thalassemia. forming inclusion bodies that damage RBCs and shorten their lifespan (see Higgs 2013). The deletion of all four a-globin genes is usually THE a-THALASSEMIAS fatal during late pregnancy or shortly after birth. This condition is called hydrops fetalis with Hb In contrast to the b-thalassemias, which are Bart’s. Hb Bart’s is a tetramer of four g-globin usually caused by point mutations of the b-glo- subunits and is ineffective as an oxygen trans- bin gene, the a-thalassemia syndromes are usu- porter: it has a very high oxygen affinity, similar ally caused by the deletion of one or more a- to that of , and does not release ox- globin genes and are subclassified according to ygen to the tissues under physiologic condi- the number of a-globin genes that are deleted tions. Therefore, the infant, whose RBCs lack (or mutated): one gene deleted (aþ-thalasse- HbF or HbA and contain mostly Hb Bart’s, suf- mia); two genes deleted on the same chromo- fers severe resulting in hydrops fetalis. some or in cis (a0-thalassemia); three genes Rare cases have been rescued by intrauterine deleted (HbH disease); or four genes deleted transfusions, but these children subsequently (hydrops fetalis with Hb Bart’s). Nondeletion require lifelong transfusion support similar to forms of a-thalassemia have also been charac- that required by children with b-thalassemia terized but are relatively uncommon. These dis- major. orders are discussed in detail in Higgs (2013). One form of nondeletion a-thalassemia, Clinically, the deletion of only one of the called Hb Constant Spring (HbCS), is particu- four a-globin genes is not associated with larly prevalent in . It is caused by a significant hematologic abnormalities and is chain termination , whichresults inthe sometimes called the “silent carrier” state for synthesis of an elongated a-globin subunit that a-thalassemia. The deletion of two a-globin accumulates at very low levels in RBCs of affect- www.perspectivesinmedicine.org genes can occur in two forms: (1) on the same ed individuals. When coinherited with the a0- chromosomes, or in cis; or (2) on opposite chro- cis deletion on the other chromosome, there re- mosomes, or in trans, which is the homozygous sults a form of HbH disease that is more severe state for the singlegene deletion, or homozygous than the typical HbH disease associated with aþ-thalassemia. The cis genotype is particular- full deletion of three a-globin genes (see Higgs ly common in Asian populations, whereas the 2013). trans genotype is highly prevalent in persons of In addition to these forms of a-thalassemia black/African ancestry. The clinical phenotype inherited in a pattern consistent with Mendelian is similar with both genotypes and consists genetics, there are two a-thalassemia syndromes of mild hypochromic, microcytic anemia, with- that are caused by de novo or acquired muta- out hemolysis, somewhat analogous to that of tions affecting expression of the a-globin genes: b-thalasssemia trait, but somewhat less severe. (1) the a-thalassemia with mental retardation Thedeletion(ormarkedlydecreasedexpression) syndrome (ATR); and (2) acquired a-thalasse- of three a-globin genes is associated with the mia (HbH disease) associated with myelodys- syndrome of HbH disease, a compensated he- plastic syndromes (ATMDS). These syndromes molytic anemia that usually does not require are described in detail in Gibbons (2012).

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B.G. Forget and H.F. Bunn

THE a-THALASSEMIA WITH MENTAL of HbAS is much higher and can reach over 30% RETARDATION SYNDROME in some populations because of survival advan- tage of HbAS heterozygotes from complica- There are two subtypes of this syndrome: (1) tions of falciparum . RBCs of persons one is associated with very large deletions in- with HbAS typically have 40% HbS and 56%– volving the a-globin genes and adjacent genes 58% HbA. Individuals with HbAS are typically on chromosome 16, that are not inherited from asymptomatic; severe hypoxia is required for a parent and appear to have arisen de novo dur- them to experience manifestations of sickle cell ing embryogenesis (the ATR-16 syndrome); and disease, called sickling. (2) the other is associated with structurally nor- The basis of sickling in patients homozygous mal a-globin genes and is caused by germline for the disorder, called sickle cell anemia or mutations in a transcription factor gene located HbSS, is polymerization of deoxy-HbS resulting on the X chromosome (the ATR-X syndrome). in the formation of multistranded fibers that The encoded transcription factor has been create a gel and change the shape of RBCs called ATRX and is an important regulator of from biconcave discs to elongated crescents. a-globin gene expression. The polymerization/sickling reaction is revers- ible following reoxygenetion of the hemoglobin. ACQUIRED a-THALASSEMIA (HbH DISEASE) Thus, an RBC can undergo repeated cycles of ASSOCIATED WITH MYELODYSPLASTIC sickling and unsickling. There are two major SYNDROMES (MDS) pathophysiological consequences of sickling:

A small number of patients with MDS, a clonal 1. Repeated cycles of sickling damage the red bone marrow disorder characterized by dis- blood cell membrane leading to abnormali- turbed hematopoiesis with cytopenias and ab- ties of permeability and cellular dehydration, normal myeloid differentiation, develop RBC eventually causing premature destruction of abnormalities consistent with acquired HbH RBCs and a chronic hemolytic anemia. disease. The a-globin genes in such patients are structurally normal, but their expression is 2. Sickled RBCs are rigid, increase blood viscos- severely impaired. The molecular basis for the ity and obstruct capillary flow, causing tissue decreased a-globin gene expression in this con- hypoxia and, if prolonged, cell death, tissue dition has been determined to be acquired necrosis/infarction, and progressive organ somatic mutations of the ATRX gene, the same damage. Acute episodes are often called gene that is mutated in the syndrome of a-thal- vaso-occlusive pain crises. assemia with mental retardation. The impair- www.perspectivesinmedicine.org ment of a-globin gene expression is more severe The pathophysiology and clinical manifesta- in the ATMDS syndrome than in the ATRX syn- tions of sickle cell disease are described in detail drome. in Schechter and Elion (2013) and Serjeant (2013). There can be a great deal of variability in the severity of the clinical manifestations of QUALITATIVE DISORDERS OF the various sickle cell syndromes in patients GLOBIN STRUCTURE with the same b-globin gene genotype and even between siblings in the same family. This Sickle Cell Disorders variability can be caused by the coinheritance of Sickle hemoglobin (HbS) results from an ami- a number of different genetic modifiers of the no acid substitution at the sixth residue of disease, including coinheritance of a-thalasse- the b-globin subunit: b6-Glu ! Val. Approxi- mia, quantitative trait loci affecting the level of mately 8% of African Americans are heterozy- production of fetal hemoglobin and other gous for this hemoglobin variant, a condition traits, as discussed in Lettre (2012). In general, called sickle cell trait or HbAS. In equatorial the clinical severity of the different sickle cell Africa, where malaria is endemic, the prevalence syndromes correlates directly with the amount

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Classification of Hemoglobin Disorders

and concentration of HbS in the red cell. The does not participate at all in polymer forma- kinetics of sickling are markedly concentration tion, this is sufficient to prevent sickling of all dependent, the speed of the reaction being in- RBCs under physiologic conditions. This situa- versely proportional to about the 30th power of tion is in contrast to what occurs when variable the starting HbS concentration. Thus, a small amounts of HbF are present in HbSS disease or increase in intracellular HbS concentration, the sickle/b-thalassemia syndromes. In the lat- such as that resulting from loss of intracellular ter disorders, the HbF is distributed in a hetero- water and cellular dehydration, can have a major cellular fashion, in which the HbF is present effect on the speed of the polymerization reac- in only a subset of cells called F cells, leaving tion. The amount and type of other non-S he- the other cells that lack HbF unprotected from moglobins in the red cell can also influence the the antisickling effects of HbF. Nevertheless, a extent or rate of sickling, and thus clinical se- marked increase in the proportion of F cells in verity. HbSC disease is associated with signifi- such cases can have a beneficial effect on the cant clinical manifestations. Why do SC pa- clinical manifestation of these sickling disor- tients often have vaso-occlusive manifestations ders. Therefore, there is a great deal of interest as dramatic as those with SS disease, whereas and ongoing research to develop strategies for AS individuals have virtually no morbidity? enhancing HbF synthesis and F-cell production HbC and HbA copolymerize to an identical de- as a therapeutic modality for sickling disorders gree with HbS (Bunn et al. 1982). Two indepen- (see Sankaran and Orkin 2013). dent factors conspire to make HbSC a disease. The presence of HbC in the red cell greatly en- UNSTABLE HEMOGLOBIN VARIANTS hances potassium efflux and red cell dehydra- tion, which increases corpuscular hemoglobin A substantial minority of Hb mutants have sub- concentration (Bunn et al. 1982) and promotes stitutionsthat alter the solubilityof the molecule polymerization. Sickling in SC patients is fur- in the red cell. The intraerythrocytic precipitated ther enhanced by the fact that they have 50% materialderived from the unstable abnormal Hb HbS, whereas HbAS individuals have 40% is detectable by a supravital stain as dark globu- HbS. This difference is because a-globin sub- lar aggregates called Heinz bodies (HBs). These units bind more readily to negatively charged intracellular inclusions reduce the life span of bA-subunits than to positively charged bC-sub- the red cell and generate a hemolytic process of units, as explained further at the end of this varied severity called congenital Heinz body he- work. Other clinically significant sickle cell syn- molytic anemia. When a red cell hemolysate of dromes include: sickle/b0-thalassemia, sickle/ an affected individual is heated to 508Cortreat- www.perspectivesinmedicine.org bþ-thalassemia, HbSD disease, and HbSO- ed with 17% isopropanol, a precipitate usually Arab disease. HbD and HbO-Arab participate develops. The hemoglobin electrophoresis often more readily than HbA in copolymer formation reveals an abnormal banding pattern. Defini- with HbS. tive diagnosis requires analysis of either globin A striking example of how another hemo- structure or DNA sequence. For more informa- globin can dramatically influence sickling is the tion on congenital Heinz body hemolytic ane- condition that results from coinheritance of mia and other hemoglobin variants summa- HbS with hereditary persistence of fetal hemo- rized in this section, see Thom et al. (2013). globin: HbSF or S/HFPH. This condition, in which there is 70% HbS and 30% HbF, HEMOGLOBIN VARIANTS WITH is clinically asymptomatic and is a notable ex- ALTERED OXYGEN AFFINITY ception to the general rule that clinical severity is directly related to the amount (or %) of HbS Over 25 hemoglobin variants have been en- in the RBC. In this condition, the HbF is dis- countered in individuals with erythrocytosis. tributed in a pancellular fashion so that every substitutions cause an increase in RBC contains 30% HbF and, because HbF oxygen affinity usually because of impairment

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B.G. Forget and H.F. Bunn

in the stability of the T or “deoxy” quaternary it is stabilized as Fe3þ. Thus, the M hemoglobins structure. Erythrocytosis is usually familial and include a58 His ! Tyr, a87 His ! Tyr, b63 is the only well-documented clinical finding. His ! Tyr, b92 His ! Tyr, g63 His ! Tyr, Increased oxygen affinity results in a decrease and g92 His ! Tyr. In addition, b67 Val ! in hemoglobin’s ability to release oxygen during Glu results in very similar biochemical features blood flow in the microcirculation. Unlike in- and clinical presentation. Affected individuals dividuals with secondary erythrocytosis caused have cyanosis similar in hue to those with meth- by high altitude exposure or to cardiac or pul- emoglobinemia in which the heme iron in nor- monary hypoxemia, those having hemoglobin mal hemoglobin has been oxidized to Fe3þ. with high oxygen affinity have normal arterial They have either normal hemoglobin levels oxygen tension. Therefore, they are not hypox- or mild anemia and no clinical manifestations emic. However, the reduction in unloading of other than their skin color. oxygen to tissues results in cellular hypoxia. Thus, the cells in the kidney that produce eryth- ropoietin sense hypoxia and up-regulate Epo IMPACT OF SUBUNIT ASSEMBLY ON gene expression leading to expansion of the HEMOGLOBIN PHENOTYPE red cell mass. About half of the high-affinity The composition of the normal and mutant he- variants can be detected by hemoglobin electro- moglobins in red cells of heterozygotes provides phoresis. Definitive diagnosis is established by insight into the assembly of human hemoglo- demonstration of a “shift to the left” in the oxy- bins (Bunn and McDonald 1983). The great ma- hemoglobin binding curve. jority of b-subunit mutants are synthesized at Less often, individuals and their relatives the same rate as bA and have normal stability. will present with low O2 saturation and cyanosis Therefore, heterozygotes would be expected to owing to a hemoglobin variant with a marked have equal amounts of normal and mutant he- reduction in oxygen affinity. The variants that moglobin. However, measurements of the pro- have been most thoroughly characterized entail portion of normal and abnormal hemoglo- amino acid replacements that destabilize the R bins in heterezygotes have revealed unexpected or “oxy” quarternary structure. Affected indi- variability. Figure 1 shows a comparison of 105 viduals have normal hemoglobin levels and no stable b-subunit mutants. Positively charged clinical manifestations other than the dusky mutants, such as hemoglobins S, C, D-Los An- skin color. geles, and E5, constitute significantly less than half of the total hemoglobin in heterozygotes and are reduced further in the presence of a-

www.perspectivesinmedicine.org THE M HEMOGLOBINS thalassemia, because of increased competition Rare families have attracted medical attention between normal and mutant b-subunits for lim- because affected individuals have cyanosis ow- iting amounts of a-subunits. In contrast, many ing to the inheritance of a hemoglobin variant of the negatively charged mutants are present in in which an amino acid substitution within the amounts exceeding that of HbA. In two hetero- heme pocket results in stabilization of the heme 3þ zygotes who had a negatively charged mutant iron in the oxidized Fe form. As discussed in (HbJ-Baltimore or HbJ-Iran) in conjunction Schechter (2013), among the most highly con- with a-thalassemia, the proportion of the mu- served amino acids within hemoglobin sub- tant hemoglobin was further increased because units are two histidines, one at helix position these bJ-subunits out-compete normal bA-sub- F8, which binds to the heme iron and one at units for limiting amounts of a-subunits. E7 in the pocket where oxygen and other ligands bind to heme. Replacement of either of these 5 histidines by tyrosine in either the a-, b-, or Levels of HbE in AE heterozygotes are lower than the others because the synthesis of bE-globin is impaired owing g-globin subunit results in an alteration in the to a defect in mRNA splicing (see Fucharoen and Weatherall electronic environment of the heme iron so that 2012 and Thein 2013).

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Classification of Hemoglobin Disorders

AB70

60 J-Balt

N-Balt 50 J-Iran

Korle-Bu C O-Arab S 40 D-Los Ang

30 E % of total hemoglobin

20 S-Oman

10 2 1 0 –1 –2 αα/αα αα/α– α–/α– α–/ – – αα/ ––

Figure 1. Impact of surface charge on hemoglobin assembly. (A) Effect of charge on the proportion of abnormal hemoglobin in individuals heterozygous for 105 stable b-globin variants. Each data point represents a mean value for a given variant. Substitutions involving a histidine residue were scored as a change of one half charge. The “21” group differs significantly from the “þ1” group (P , 0.001). (B) Effect of a-thalassemia on the proportion of seven positively charged b-subunit variants (blue) and three negatively charged variants (red). (Updated from Bunn and McDonald 1983 and Bunn 1987.)

Analyses of the proportion of b-subunit mutant Accordingly, in red cells of heterozygotes, it is in heterozygotes suggest that alterations in sur- present in much less abundance than HbA. facechargecontributetodifferentratesofassem- However, because of its markedly enhanced bly of the hemoglobin tetramer. This hypothesis polymerization, heterozygotes with only 20% issupportedby invitromixing experiments with HbS-Oman and one-gene deletion a-thalas- normal and mutant subunits showing that when semia (a-/aa) have hemolytic anemia and signs a-subunits are present in limiting amounts and symptoms of sickle vaso-occlusion. In con- www.perspectivesinmedicine.org (mimicking a-thalassemia), negatively charged trast, HbS-Oman heterozygotes with more mutants are formed much more readily than are severe a-thalassemia (a-/a-) have only 13% positively charged mutants (Mrabet et al. 1986; HbS-Oman and a normal clinical and hemato- Adachi et al. 1998). logic phenotype (Nagel et al. 1998). This electrostatic model of hemoglobin as- The impact of surface charge on hemoglo- sembly has clinical implications. Differences bin assembly also explains the differences in the in rates of assembly explain the higher propor- levels of HbA2 (Bunn 1987) and HbF (Adams tion in HbS in sickle C (SC) disease than in sickle et al. 1985; Chui et al. 1990) that accompany trait (AS), a difference that contributes signif- certain hematological disorders. For example, icantly to HbS polymerization and consequent a-hemoglobin subunits bind more readily to vaso-occlusion in SC patients (Bunn et al. 1982). b than to the positively charged d. Accordingly, HbS-Oman is a rare stable mutant with both HbA2 levels are decreased in the presence the sickle (b6Glu ! Val) substitution and of limiting levels of a-hemoglobin, e.g., a- b121Glu ! Lys, a substitution that stabilizes thalassemia as well as iron deficiency in which the sickle polymer. Thus, b-subunits of HbS- there is an acquired impairment of a-globin Oman have a gain of three positive charges. synthesis.

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B.G. Forget and H.F. Bunn

POSTTRANSLATIONAL ALTERATIONS acetylated at the amino terminus. These post- IN HEMOGLOBIN STRUCTURE translational modifications are highly depen- dent on the sequence of neighboring amino Awide range of posttranslational modifications acids. All human undergo cleavage of of hemoglobin structure have been identified, the amino-terminal methionine but none un- some of which result in significant perturba- dergo amino-terminal acetylation except for tions of function. 20% of g-globin subunits (HbFI). However, rare human hemoglobin variants have been Nonenzymatic Glycation identified that have amino acid replacements in the amino-terminal region that cause both The concentration of glucose in the RBC cytosol retention of the initiator methionine and N- is nearly that of the plasma. Glucose is in equi- acetylation. librium between the cyclic hemiacetal structure and the open aldehyde structure. The latter is able to react covalently with amino groups of a Amino-Terminal Carbamylation wide range of . Because this reaction Urea in the plasma is in equilibrium with am- favors amino groups with relatively low pKas, monium and cyanate ions. Cyanate is able to the most abundant is at bind covalently to the amino termini of a- and the amino termini of a- and b-globin. In nor- b-globin subunits to form adducts. Carbamy- mal red cells, 5% of hemoglobin has a glucose lated hemoglobin is not detectable in normal adduct at the b-amino terminus designated red cells but can accumulate to measurable lev- HbAIc: els in patients with uremia.

+ β HC=O HC=N–β CH2–N H2– HCOH HCOH C=O Deamidation HOCH HOCH HOCH β –NH2 + HCOH 1 HCOH 2 HCOH In many proteins, the amino group on certain HCOH HCOH HCOH asparagine residues, particularly when adjacent CH OH CH2OH CH2OH 2 to a histidine, dissociates leaving aspartic acid. Glucose Aldimine Ketoamine Deamidation does not occur on asparagines in normal globin subunits. However, seven human Reaction 1 shows the formation of the Schiff variants have been identified in which partial or base or aldimine adduct. This product then re- complete deamidation occurs. Four of these en- arranges to the more stable (virtually irrever- tail replacements by asparagine. The deamida- www.perspectivesinmedicine.org sible) ketoamine linkage (reaction 2). HbAIc tion does not appear to impact function in any and other less abundant glucose adducts are of these variants. formed in a linear manner over the 120-day life span of the red cell. Therefore, HbAIc levels are low in individuals with hemolysis. Because the ACQUIRED ALTERATIONS IN level of HbAIc is an accurate reflection of the HEMOGLOBIN FUNCTION average concentration of plasma glucose over Methemoglobinemia the preceding 2 months, it has proven to be very useful in monitoring therapeutic control Certain drugs and toxins can cause enhanced in patients with diabetes. oxidation of heme iron to form methemo- globin. Above levels of 10%, individuals appear cyanotic. The extent of he- Amino-Terminal Acetylation moglobin oxidation is determined by spectro- Shortly following translation of most proteins, photometric analysis of the blood. Methemo- the amino-terminal (initiator) methionine is globinemia impairs oxygenation of tissues cleaved and often the adjacent amino acid is because the Fe3þ heme is incapable of binding

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Classification of Hemoglobin Disorders

to oxygen, and, even more importantly, the bin binding curve “to the right.” Except under presence of one or two oxidized hemes per tet- clinical circumstances resulting in either acido- ramer stabilizes the R quaternary structure and sis or alkalosis, plasma pH and red cell pH are thereby increases the affinity of the remaining tightly regulated at 7.4 and 7.2, respectively. In hemes for oxygen. Central nervous and cardio- contrast, levels of red cell 2,3-BPG can vary con- vascular symptoms and signs begin to appear siderably in a wide range of clinical settings. when methemoglobin exceeds 30%. Much low- Patients with low levels, such as those who er levels are seen in individuals with congenit- have received large amounts of stored RBCs al methemoglobinemia owing to deficiency in will have increased oxygen affinity, which could cytochrome b5 reductase. Both toxic and con- compromise tissue oxygenation. Increased lev- genital methemoglobinemia can be effectively els of RBC 2,3-BPG are much more commonly treated by administration of the redox dye meth- observed, primarily in patients with some form ylene blue. of hypoxia. As a result, the “right shift” in the oxyhemoglobin dissociation curve significantly enhances the unloading of oxygen to tissues and Carboxyhemoglobinemia is therefore a major mode of compensation, (CO) binds to heme iron of particularly in patients with severe anemia. hemoglobin with an affinity 210 greater than that of oxygen. Normal red cells contain a very small amount of owing to the re- REFERENCES lease of CO from macrophages following the up- Reference is also in this collection. take of senescent red cells and degradation of Adachi K, Yamaguchi T, Pang J, Surrey S. 1998. Effects of heme by . Higher, albeit nontox- increased anionic charge in the b-globin chain on assem- ic, levels of HbCO are present in patients with bly of hemoglobin in vitro. Blood 91: 1438–1445. hemolytic anemias. Inhalation of CO from in- Adams JD, Coleman MB, Hayes J, Morrison WT, Stein- complete combustion of hydrocarbons can result berg MH. 1985. Modulation of fetal hemoglobin synthesis by iron deficiency. N Engl J Med 313: 1402– in toxic carboxyhemoglobinemia. As in methe- 1405. moglobinemia, CO poisoning causes morbidity Bunn HF. 1987. Subunit assembly of hemoglobin: An im- and mortality by ischemia owing to a left shift portant determinant of hematologic phenotype. Blood in the oxyhemoglobin binding curve, thereby 69: 1–6. Bunn HF, McDonald MJ. 1983. Electrostatic interactions in impairing the unloading of oxygen to cells and the assembly of haemoglobin. Nature 306: 498–500. tissues. Patients with acute CO poisoning gen- Bunn HF, Noguchi CT, Hofrichter J, Schechter GP, erally develop CNS and cardiovascular symp- Schechter AN, Eaton WA. 1982. Molecular and cellular www.perspectivesinmedicine.org toms when levels exceed 20%. Levels of 40%– pathogenesis of hemoglobin SC disease. Proc Natl Acad Sci 79: 7527–7531. 60% HbCO result in stupor, coma, and death. All Chui DH, Patterson M, Dowling CE, Kazazian HJ, Ken- symptomatic patients should be promptly re- dall AG. 1990. Hemoglobin Bart’s disease in an Italian moved from further exposure to CO and treated boy. Interaction between a-thalassemia and hereditary with oxygen. If possible, those with severe toxic- persistence of fetal hemoglobin. N Engl J Med 323: 179–182. ity should be treated with hyperbaric oxygen Dailey HA, Meissner PN. 2013. Erythroid heme biosynthesis administration or exchange transfusion. and its disorders. Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a011676. Feng L, Gell DA, Zhou S, Gu L, Kong Y, Li J, Hu M, Yan N, Alterations in Oxygen Affinity Lee C, Rich AM, et al. 2004. Molecular mechanism of AHSP-mediated stabilization of a-hemoglobin. Cell 119: þ As mentioned above, protons (H ) and 2,3- 629–640. BPG are potent allosteric modulators of oxygen Fucharoen S, Weatherall DJ. 2012. The thal- affinity in normal RBC. Because of their prefer- assemias. Cold Spring Harb Perspect Med 2: a011734. Higgs DR. 2013. The molecular basis of a-thalassemia. ential binding to the T or “deoxy” quaternary Cold Spring Harb Perspect Med 3: a011718. structure of hemoglobin, they lower the affinity Kihm AJ, Kong Y, Hong W, Russell JE, Rouda S, Adachi K, for oxygen and therefore shift the oxyhemoglo- Simon MC, Blobel GA, Weiss MJ. 2002. An abundant

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B.G. Forget and H.F. Bunn

erythroid protein that stabilizes free a-haemoglobin. Na- 1998. HbS-Oman heterozygote: A new dominant sickle ture 417: 758–763. syndrome. Blood 92: 4375–4382. Lettre G. 2012. The search for genetic modifiers of disease Nienhuis AW,Nathan DG. 2012. Pathophysiology and clinical severity in the b-. Cold Spring Harb manifestations of the b-thalassemias. Cold Spring Harb Perspect Med 2: a015032. Perspect Med 2: a011726. Mollan TL, Yu X, Weiss MJ, Olson JS. 2010. The role of a- Sankaran VG, Orkin SH. 2013. The switch from fetal to hemoglobin stabilizing protein in redox chemistry, dena- adult hemoglobin. Cold Spring Harb Perspect Med 3: turation, and hemoglobin assembly. Antioxid Redox Sig- a011643. nal 12: 219–231. Schechter AN. 2013. Hemoglobin function. Cold Spring Mollan TL, Khandros E, Weiss MJ, Olson JS. 2012. The Harb Perspect Med 3: a011650. kinetics of a-globin binding to a hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of a Schechter AN, Elion J. 2013. Pathophysiology of sickle cell hemichrome folding intermediate. J Biol Chem 287: disease. Cold Spring Harb Perspect Med (to be published). 11338–11350. Serjeant G. 2013. Natural history of sickle cell disease. Mrabet NT, McDonald MJ, Turci S, Sarkar R, Szabo A, Cold Spring Harb Perspect Med doi: 10.1101/cshperspect. Bunn HF.1986. Electrostatic attraction governs the dimer a011783. assembly of human hemoglobin. J Biol Chem 261: 5222– Thein SL. 2013. Molecular basis of b-thalassemia. Cold 5228. Spring Harb Perspect Med doi: 10.1101/cshperspect. Musallam KM, Tahar AT,Rachmilewitz EA. 2012. b-Thalas- a011700. semia intermedia: A clinical perspective. Cold Spring Thom CS, Dickson CF, Gell DA, Weiss MJ. 2013. Hemoglo- Harb Perspect Med 2: a013482. bin variants: Biochemical properties and clinical corre- Nagel RL, Daar S, Romero JR, Suzuka SM, Gravell D, lates. Cold Spring Harb Perspect Med doi: 10.1101/cshper- Bouhassira E, Schwartz RS, Fabry M, Krishnamoorthy R. spect.a011858. www.perspectivesinmedicine.org

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Classification of the Disorders of Hemoglobin

Bernard G. Forget and H. Franklin Bunn

Cold Spring Harb Perspect Med 2013; doi: 10.1101/cshperspect.a011684

Subject Collection Hemoglobin and Its Diseases

The Natural History of Sickle Cell Disease Transcriptional Mechanisms Underlying Graham R. Serjeant Hemoglobin Synthesis Koichi R. Katsumura, Andrew W. DeVilbiss, Nathaniel J. Pope, et al. Current Management of Sickle Cell Anemia Iron Deficiency Anemia: A Common and Curable Patrick T. McGann, Alecia C. Nero and Russell E. Disease Ware Jeffery L. Miller Cell-Free Hemoglobin and Its Scavenger Proteins: Management of the Thalassemias New Disease Models Leading the Way to Targeted Nancy F. Olivieri and Gary M. Brittenham Therapies Dominik J. Schaer and Paul W. Buehler Clinical Manifestations of α-Thalassemia The Molecular Basis of β-Thalassemia Elliott P. Vichinsky Swee Lay Thein Erythroid Heme Biosynthesis and Its Disorders Erythropoiesis: Development and Differentiation Harry A. Dailey and Peter N. Meissner Elaine Dzierzak and Sjaak Philipsen Hemoglobin Variants: Biochemical Properties and Erythropoietin Clinical Correlates H. Franklin Bunn Christopher S. Thom, Claire F. Dickson, David A. Gell, et al. The Prevention of Thalassemia Classification of the Disorders of Hemoglobin Antonio Cao and Yuet Wai Kan Bernard G. Forget and H. Franklin Bunn The Switch from Fetal to Adult Hemoglobin The Molecular Basis of α-Thalassemia Vijay G. Sankaran and Stuart H. Orkin Douglas R. Higgs

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