classification of the disorders of hemoglobin

2013; doi: 10.1101/cshperspect.a011684Cold Spring Harb Perspect Med Bernard G. Forget and H. Franklin Bunn Classification of the Disorders of Hemoglobin

Subject Collection Hemoglobin and Its Diseases

The Natural History of Sickle Cell DiseaseGraham R. Serjeant Hemoglobin Synthesis

Transcriptional Mechanisms Underlying

Nathaniel J. Pope, et al.Koichi R. Katsumura, Andrew W. DeVilbiss,

Current Management of Sickle Cell Anemia

WarePatrick T. McGann, Alecia C. Nero and Russell E. Disease

Iron Deficiency Anemia: A Common and Curable

Jeffery L. Miller

TherapiesTargetedNew Disease Models Leading the Way to

Cell-Free Hemoglobin and Its Scavenger Proteins:

Dominik J. Schaer and Paul W. Buehler

Management of the ThalassemiasNancy F. Olivieri and Gary M. Brittenham

-ThalassemiaαClinical Manifestations of Elliott P. Vichinsky

-ThalassemiaβThe Molecular Basis of Swee Lay Thein

Erythroid Heme Biosynthesis and Its DisordersHarry A. Dailey and Peter N. Meissner

Erythropoiesis: Development and DifferentiationElaine Dzierzak and Sjaak Philipsen

Clinical CorrelatesHemoglobin Variants: Biochemical Properties and

Gell, et al.Christopher S. Thom, Claire F. Dickson, David A.

ErythropoietinH. Franklin Bunn

The Prevention of ThalassemiaAntonio Cao and Yuet Wai Kan

Classification of the Disorders of HemoglobinBernard G. Forget and H. Franklin Bunn

The Switch from Fetal to Adult HemoglobinVijay G. Sankaran and Stuart H. Orkin

-ThalassemiaαThe Molecular Basis of Douglas R. Higgs

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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Classification of the Disordersof Hemoglobin

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 MedicalSchool, Boston, Massachusetts 02115

Correspondence: [email protected]

Over the years, study of the disorders of hemoglobin has served as a paradigm for gaininginsights into the cellular and molecular biology, as well as the pathophysiology, of inheritedgenetic disorders. To date, more than 1000 disorders of hemoglobin synthesis and/or struc-ture have been identified and characterized. Study of these disorders has establishedthe principle of how a mutant genotype can alter the function of the encoded protein,which in turn can lead to a distinct clinical phenotype. Genotype/phenotype correlationshave 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 theclassification.

An impressive degree of molecular “engineer-ing” was necessary for the evolution of a

multisubunit protein that higher organisms re-quire for optimal oxygen homeostasis and buf-fering of acidic metabolic waste products. Eachglobin subunit must form a stable linkage withheme (ferroprotoporphyrin IX) situated on theexternal surface of the protein so that oxygenin the RBC cytosol can bind reversibly to thehemes’ iron atoms. Moreover, the hydrophobiccleft into which the heme is inserted must beable to protect the Fe2þ heme iron from oxida-

tion to Fe3þ, which is incapable of binding ox-ygen.3 Delicate noncovalent interactions be-tween unlike globin subunits are required forthe hemoglobin tetramer a2b2 to bind and un-load oxygen in a cooperative manner, therebyassuring maximal transport to actively metab-olizing cells. This phenomenon is reflected bya sigmoid oxygen-binding curve that dependson hemoglobin tetramer having two quaternarystructures: the T or deoxy conformer that haslow oxygen affinity and the R or oxy conformerthat 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

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tuning of hemoglobin function comes from itsallosteric behavior triggered by the binding oftwo small effector molecules 2,3-BPG, and pro-tons to specific sites on the T structure distantfrom the heme groups.4 To endow the bloodwith high oxygen carrying capacity hemoglo-bin must be stuffed into flexible circulatingRBCs. A remarkably high degree of solubilityis required for hemoglobin to achieve an intra-cellular concentration of �34 g/dl or 5 mM

(tetramer).To reach such a high corpuscular hemoglo-

bin concentration it is essential that a-globin aswell as b-globin (or g-globin) mRNA be ex-pressed at very high levels during erythroid dif-ferentiation. Moreover, a- and non-a-globinsynthesis must be closely matched. As explainedin Nienhuis and Nathan (2012), subunit imbal-ance is central to the pathophysiologyof the thal-assemias. Freea-globin subunits are particularlytoxic to erythroid cells. This threat is alleviatedby the presence of a-hemoglobin stabilizingprotein (AHSP), a chaperone that is expressedat high levels in erythroid cells and binds specif-ically and tightly to heme-intact a-globin sub-units (Kihm et al. 2002; Feng et al. 2004; Mollanet al. 2010, 2012). The AHSP protects the cellfrom potentially toxic oxidized (Fe3þ) heme un-til it is reduced to functional Fe2þ heme by cy-tochrome b5 reductase. On encountering a freeheme-intact b-globin subunit, the a-globindissociates from AHSP to form the extremelystable ab dimer. As discussed later in thiswork, this process is facilitated by electrostaticattraction between positively charged a-globinsubunits and negatively charged b-globin sub-units.

In view of the multiple molecular con-straints that are required for high-level produc-tion of fully functional and highly soluble he-moglobin, it is no surprise that Murphy’s lawis in full force: whatever can go wrong will.As discussed briefly here, and in much moredetail in Thein (2013), Higgs (2013), Nienhuisand Nathan (2012), and Musallam et al. (2012),

mutations of globin genes that impair synthesisgive rise to thalassemia and anemia of varyingdegree. In addition, well-defined clinical andhematologic phenotypes are associated withmutations that alter the structure of globin sub-units, discussed in more detail in Thom et al.(2013). Impairment of hemoglobin solubilitycan be caused either by the formation of intra-cellular polymers (sickle cell disease) or by thedevelopment of amorphous precipitates (con-genital Heinz body hemolytic anemia). Abnor-malities of oxygen binding can lead either toerythrocytosis (high O2 affinity mutants) or tocyanosis (low O2 affinity mutants). Some glo-bin mutants have structural alterations withinthe heme pocket that result in oxidation ofthe heme iron and pseudocyanosis because ofmethemoglobinemia.

GENERAL CLASSIFICATION OFHEMOGLOBIN DISORDERS

Hemoglobin disorders can be broadly classifiedinto two general categories (as listed in Table 1):

1. Those in which there is a quantitative defectin the production of one of the globin sub-units, either total absence or marked reduc-tion. These are called the thalassemia syn-dromes.

2. Those in which there is a structural defect inone of the globin subunits.

The majority of human hemoglobin mu-tants were discovered as an incidental finding,unassociated with any hematologic or clinicalphenotype. However, a number ofa- andb-glo-bin mutants are associated with distinct clinicalphenotypes. These fall into five broad catego-ries: the sickle syndromes (SS, SC, Sb0-thalasse-mia, and Sbþ-thalassemia); unstable mutantscausing congenital Heinz body hemolytic ane-mia; mutants with high oxygen affinity result-ing in erythrocytosis; low oxygen affinity mu-tants and the M hemoglobins causing cyanosis;and mutants associated with a thalassemia phe-notype. Most of these disorders are inheritedgenetic defects, but there are some defects thatare acquired or occur de novo.

4For more detailed information on hemoglobin function,see Schechter (2013).

B.G. Forget and H.F. Bunn

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THE THALASSEMIA SYNDROMES

The thalassemia syndromes are inherited dis-orders characterized by absence or markedlydecreased accumulation of one of the globin

subunits of hemoglobin. In the alpha (a)-thal-assemias, there is absent or decreased pro-duction of a-globin subunits, whereas in thebeta (b)-thalassemias, there is absent or re-duced production of b-globin subunits. Rare

Table 1. Classification of hemoglobin disorders

I. QUANTITATIVE DISORDERS OF GLOBINCHAIN SYNTHESIS/ACCUMULATION

The thalassemia syndromesA. b-ThalassemiaClinical classification:b-Thalassemia minor or traitb-Thalassemia majorb-Thalassemia intermedia

Biochemical/genetic classification:

b0-Thalassemia

bþ-Thalassemia

d-Thalassemia

g-Thalassemia

Lepore fusion gene

db-Thalassemia

1gdb-Thalassemia

HPFH

“Dominant” b-thalassemia (structural variants with

b-thalassemia phenotype)

b-Thalassemia with other variants:

HbS/b-thalassemia

HbE/b-thalassemia

Other

B. a-Thalassemia

Deletions of a-globin genes:

One gene: aþ-thalassemia

Two genes in cis: a0-thalassemia

Two genes in trans: homozygous aþ-thalassemia

(phenotype of a0-thalassemia)

Three genes: HbH disease

Four genes: Hydrops fetalis with Hb Bart’s

Nondeletion mutants:

Hb Constant Spring

Other

C. De novo and acquired a-thalassemia

a-Thalassemia with mental retardation syndrome (ATR):

Due to large deletions on chromosome 16 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

II. QUALITATIVE DISORDERS OF GLOBINSTRUCTURE: STRUCTURAL VARIANTS OFHEMOGLOBIN

A. Sickle cell disordersSA, sickle cell traitSS, sickle cell anemia/diseaseSC, HbSC diseaseS/b thal, sickle b-thalassemia diseaseS with other Hb variants: D, O-Arab, otherSF, Hb S/HPFH

B. Hemoglobins with decreased stability

(unstable hemoglobin variants)

Mutants causing congenital Heinz body hemolytic

anemia

Acquired instability—oxidant hemolysis: Drug-

induced, G6PD deficiency

C. Hemoglobins with altered oxygen affinity

High/increased oxygen affinity states:

Fetal red cells

Decreased RBC 2,3-BPG

Carboxyhemoglobinemia, HbCO

Structural variants

Low/decreased oxygen affinity states:

Increased RBC 2,3-BPG

Structural variants

D. Methemoglobinemia

Congenital methemoglobinemia:

Structural variants

Cytochrome b5 reductase deficiency

Acquired (toxic) methemoglobinemia

E. Posttranslational modifications

Nonenzymatic glycosylation

Amino-terminal acetylation

Amino-terminal carbamylation

Deamidation

Classification of Hemoglobin Disorders

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thalassemias affecting the production of delta(d)- or gamma (g)-globin subunits have alsobeen described but are not clinically signif-icant disorders. Combined deficiency of d þb-globin subunits, or of all of the b-like glo-bin subunits also occurs. These disorders aredescribed in detail in Thein (2013) and Higgs(2013).

THE b-THALASSEMIAS

The b-thalassemias can be subclassified intothose in which there is total absence of normalb-globin subunit synthesis or accumulation,the b0-thalassemias, and those in which somestructurally normal b-globin subunits are syn-thesized, but in markedly decreased amounts,the bþ-thalassemias. The molecular basis of theb-thalassemias is very heterogeneous (see Thein2013), with over 200 different mutations havingbeen described. In general, the mutations caus-ing b-thalassemia are point mutations affect-ing a single nucleotide, or a small number ofnucleotides, in the b-globin gene. Rare deletionforms of b-thalassemia have also been de-scribed. One of these deletions is caused by “un-equal” crossing over between the linked andpartially homologous d- and b-globin genes,resulting in the formation of a fusion db-globingene, the Lepore gene, that has a low level ofexpression. Large deletions involving part orall of the b-globin gene cluster are responsiblefor the db-thalassemias, the 1gdb-thalassemias,and the hereditary persistence of fetal hemoglo-bin (HPFH) syndromes.

Despite the marked heterogeneity in themolecular basis of the b-thalassemias, the clin-ical phenotype of these disorders is relativelyhomogeneous because of their common patho-physiology: deficiency of HbA tetramers andexcess accumulation of free a-subunits incapa-ble of forming hemoglobin tetramers because ofdeficiency of b-like globin subunits (see Nien-huis and Nathan 2012). In heterozygotes (b-thalassemia trait orb-thalassemia minor), thereis mild to moderate hypochromic microcyticanemia, without evidence of hemolysis, whereasin homozygotes or compound heterozygotes

(b-thalassemia major), there is usually a severetransfusion-dependent hemolytic anemia asso-ciated with marked ineffective erythropoiesisresulting in destruction of erythroid precursorcells in the bone marrow.

A less common clinical phenotype is re-ferred to as b-thalassemia intermedia. In thisclinical disorder, there is a moderate to severe,partially compensated, hemolytic anemia thatdoes not require chronic transfusion therapyto maintain a satisfactory circulating hemo-globin level in the affected patient, althoughoccasional transfusions may be required if theanemia worsens because of associated com-plications. Such patients have a milder diseasebecause there is less severe a- to non-a-globinsubunit imbalance than in typical b-thalasse-mia major patients, resulting in less accumu-lation of free a-subunits that cause the ineffec-tive erythropoiesis. There are different possiblecauses for such a lessened a- to non-a-globinsubunit imbalance, including: inheritance ofmilder bþ-thalassemia mutations with less se-vere than usual deficiency of b-globin subunitproduction; coinheritance of a form of a-thal-assemia; or coinheritance of another genetictrait associated with increased production ofthe g-subunit of fetal hemoglobin. Most pa-tients with b-thalassemia intermedia carrytwo mutant b-globin genes: they have a geno-type typical ofb-thalasemia major, but the phe-notype is modified by one of the factors listedabove. However, rare cases of b-thalassemia in-termedia are caused by heterozygosity for asingle mutant b-globin gene associated withthe production of a highly unstable b-globinsubunit that causes RBC damage in a fashionsimilar to excess free a-subunits; this is theso-called “dominant b-thalassemia” (Thom etal. 2013).

The db-thalassemias are associated with to-tal deficiency of b-globin subunit production,but are clinically milder than typical cases ofb0-thalassemia, because there is an associated per-sistent high level of expression of the g-subunitof fetal hemoglobin that decreases the degreeof a-subunit excess. The 1gdb-thalassemias areassociated with neonatal hemolytic anemia,but this resolves during the first few months



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of life and the associated phenotype in adults isthat of b-thalassemia trait or b-thalassemia mi-nor. The syndrome of hereditary persistence offetal hemoglobin (HPFH) is not, strictly speak-ing, a form of b-thalassemia because it is notassociated with significant a- to non-a-globinsubunit imbalance, but is characterized by highlevels of persistent g-globin production and isfrequently considered within the spectrum ofdb-thalassemia.

THE a-THALASSEMIAS

In contrast to the b-thalassemias, which areusually caused by point mutations of the b-glo-bin gene, the a-thalassemia syndromes are usu-ally caused by the deletion of one or more a-globin genes and are subclassified according tothe number of a-globin genes that are deleted(or mutated): one gene deleted (aþ-thalasse-mia); two genes deleted on the same chromo-some or in cis (a0-thalassemia); three genesdeleted (HbH disease); or four genes deleted(hydrops fetalis with Hb Bart’s). Nondeletionforms of a-thalassemia have also been charac-terized but are relatively uncommon. These dis-orders are discussed in detail in Higgs (2013).

Clinically, the deletion of only one of thefour a-globin genes is not associated withsignificant hematologic abnormalities and issometimes called the “silent carrier” state fora-thalassemia. The deletion of two a-globingenes can occur in two forms: (1) on the samechromosomes, or in cis; or (2) on opposite chro-mosomes, or in trans, which is the homozygousstate for the single gene deletion, or homozygousaþ-thalassemia. The cis genotype is particular-ly common in Asian populations, whereas thetrans genotype is highly prevalent in persons ofblack/African ancestry. The clinical phenotypeis similar with both genotypes and consistsof mild hypochromic, microcytic anemia, with-out hemolysis, somewhat analogous to that ofb-thalasssemia trait, but somewhat less severe.The deletion (or markedlydecreasedexpression)of three a-globin genes is associated with thesyndrome of HbH disease, a compensated he-molytic anemia that usually does not require

treatment by RBC transfusion. The basis ofthe hemolysis is the excess accumulation of b-globin subunits that self-associate to formb-chain tetramers or HbH. In contrast to thesituation in the b-thalssemias in which excessa-globin subunits rapidly form insoluble ag-gregates, excess b-globin chains can form sol-uble tetramers (HbH). However, HbH is rela-tively unstable and does precipitate as RBCs age,forming inclusion bodies that damage RBCsand shorten their lifespan (see Higgs 2013).The deletion of all foura-globin genes is usuallyfatal during late pregnancy or shortly after birth.This condition is called hydrops fetalis with HbBart’s. Hb Bart’s is a tetramer of four g-globinsubunits and is ineffective as an oxygen trans-porter: it has a very high oxygen affinity, similarto that of myoglobin, and does not release ox-ygen to the tissues under physiologic condi-tions. Therefore, the infant, whose RBCs lackHbF or HbA and contain mostly Hb Bart’s, suf-fers severe hypoxia resulting in hydrops fetalis.Rare cases have been rescued by intrauterinetransfusions, but these children subsequentlyrequire lifelong transfusion support similar tothat required by children with b-thalassemiamajor.

One form of nondeletion a-thalassemia,called Hb Constant Spring (HbCS), is particu-larly prevalent in southeast Asia. It is caused by achain termination mutation, which results in thesynthesis of an elongated a-globin subunit thataccumulates at very low levels in RBCs of affect-ed individuals. When coinherited with the a0-cis deletion on the other chromosome, there re-sults a form of HbH disease that is more severethan the typical HbH disease associated withfull deletion of three a-globin genes (see Higgs2013).

In addition to these forms of a-thalassemiainherited in a pattern consistent with Mendeliangenetics, there are twoa-thalassemia syndromesthat are caused by de novo or acquired muta-tions affecting expression of the a-globin genes:(1) the a-thalassemia with mental retardationsyndrome (ATR); and (2) acquired a-thalasse-mia (HbH disease) associated with myelodys-plastic syndromes (ATMDS). These syndromesare described in detail in Gibbons (2012).



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THE a-THALASSEMIA WITH MENTALRETARDATION SYNDROME

There are two subtypes of this syndrome: (1)one is associated with very large deletions in-volving the a-globin genes and adjacent geneson chromosome 16, that are not inherited froma parent and appear to have arisen de novo dur-ing embryogenesis (the ATR-16 syndrome); and(2) the other is associated with structurally nor-mal a-globin genes and is caused by germlinemutations in a transcription factor gene locatedon the X chromosome (the ATR-X syndrome).The encoded transcription factor has beencalled ATRX and is an important regulator ofa-globin gene expression.

ACQUIRED a-THALASSEMIA (HbH DISEASE)ASSOCIATED WITH MYELODYSPLASTICSYNDROMES (MDS)

A small number of patients with MDS, a clonalbone marrow disorder characterized by dis-turbed hematopoiesis with cytopenias and ab-normal myeloid differentiation, develop RBCabnormalities consistent with acquired HbHdisease. The a-globin genes in such patientsare structurally normal, but their expression isseverely impaired. The molecular basis for thedecreased a-globin gene expression in this con-dition has been determined to be acquiredsomatic mutations of the ATRX gene, the samegene that is mutated in the syndrome of a-thal-assemia with mental retardation. The impair-ment ofa-globin gene expression is more severein the ATMDS syndrome than in the ATRX syn-drome.

QUALITATIVE DISORDERS OFGLOBIN STRUCTURE

Sickle Cell Disorders

Sickle hemoglobin (HbS) results from an ami-no acid substitution at the sixth residue ofthe b-globin subunit: b6-Glu ! Val. Approxi-mately 8% of African Americans are heterozy-gous for this hemoglobin variant, a conditioncalled sickle cell trait or HbAS. In equatorialAfrica, where malaria is endemic, the prevalence

of HbAS is much higher and can reach over 30%in some populations because of survival advan-tage of HbAS heterozygotes from complica-tions of falciparum malaria. RBCs of personswith HbAS typically have 40% HbS and 56%–58% HbA. Individuals with HbAS are typicallyasymptomatic; severe hypoxia is required forthem to experience manifestations of sickle celldisease, called sickling.

The basis of sickling in patients homozygousfor the disorder, called sickle cell anemia orHbSS, is polymerization of deoxy-HbS resultingin the formation of multistranded fibers thatcreate a gel and change the shape of RBCsfrom biconcave discs to elongated crescents.The polymerization/sickling reaction is revers-ible following reoxygenetion of the hemoglobin.Thus, an RBC can undergo repeated cycles ofsickling and unsickling. There are two majorpathophysiological consequences of sickling:

1. Repeated cycles of sickling damage the redblood cell membrane leading to abnormali-ties of permeability and cellular dehydration,eventually causing premature destruction ofRBCs and a chronic hemolytic anemia.

2. Sickled RBCs are rigid, increase blood viscos-ity and obstruct capillary flow, causing tissuehypoxia and, if prolonged, cell death, tissuenecrosis/infarction, and progressive organdamage. Acute episodes are often calledvaso-occlusive pain crises.

The pathophysiology and clinical manifesta-tions of sickle cell disease are described in detailin Schechter and Elion (2013) and Serjeant(2013). There can be a great deal of variabilityin the severity of the clinical manifestations ofthe various sickle cell syndromes in patientswith the same b-globin gene genotype andeven between siblings in the same family. Thisvariability can be caused by the coinheritance ofa number of different genetic modifiers of thedisease, including coinheritance of a-thalasse-mia, quantitative trait loci affecting the level ofproduction of fetal hemoglobin and othertraits, as discussed in Lettre (2012). In general,the clinical severity of the different sickle cellsyndromes correlates directly with the amount



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and concentration of HbS in the red cell. Thekinetics of sickling are markedly concentrationdependent, the speed of the reaction being in-versely proportional to about the 30th power ofthe starting HbS concentration. Thus, a smallincrease in intracellular HbS concentration,such as that resulting from loss of intracellularwater and cellular dehydration, can have a majoreffect on the speed of the polymerization reac-tion. The amount and type of other non-S he-moglobins in the red cell can also influence theextent or rate of sickling, and thus clinical se-verity. HbSC disease is associated with signifi-cant clinical manifestations. Why do SC pa-tients often have vaso-occlusive manifestationsas dramatic as those with SS disease, whereasAS individuals have virtually no morbidity?HbC and HbA copolymerize to an identical de-gree with HbS (Bunn et al. 1982). Two indepen-dent factors conspire to make HbSC a disease.The presence of HbC in the red cell greatly en-hances potassium efflux and red cell dehydra-tion, which increases corpuscular hemoglobinconcentration (Bunn et al. 1982) and promotespolymerization. Sickling in SC patients is fur-ther enhanced by the fact that they have �50%HbS, whereas HbAS individuals have �40%HbS. This difference is because a-globin sub-units bind more readily to negatively chargedbA-subunits than to positively charged bC-sub-units, as explained further at the end of thiswork. Other clinically significant sickle cell syn-dromes include: sickle/b0-thalassemia, sickle/bþ-thalassemia, HbSD disease, and HbSO-Arab disease. HbD and HbO-Arab participatemore readily than HbA in copolymer formationwith HbS.

A striking example of how another hemo-globin can dramatically influence sickling is thecondition that results from coinheritance ofHbS with hereditary persistence of fetal hemo-globin: HbSF or S/HFPH. This condition, inwhich there is �70% HbS and �30% HbF,is clinically asymptomatic and is a notable ex-ception to the general rule that clinical severityis directly related to the amount (or %) of HbSin the RBC. In this condition, the HbF is dis-tributed in a pancellular fashion so that everyRBC contains �30% HbF and, because HbF

does not participate at all in polymer forma-tion, this is sufficient to prevent sickling of allRBCs under physiologic conditions. This situa-tion is in contrast to what occurs when variableamounts of HbF are present in HbSS disease orthe sickle/b-thalassemia syndromes. In the lat-ter disorders, the HbF is distributed in a hetero-cellular fashion, in which the HbF is presentin only a subset of cells called F cells, leavingthe other cells that lack HbF unprotected fromthe antisickling effects of HbF. Nevertheless, amarked increase in the proportion of F cells insuch cases can have a beneficial effect on theclinical manifestation of these sickling disor-ders. Therefore, there is a great deal of interestand ongoing research to develop strategies forenhancing HbF synthesis and F-cell productionas a therapeutic modality for sickling disorders(see Sankaran and Orkin 2013).

UNSTABLE HEMOGLOBIN VARIANTS

A substantial minority of Hb mutants have sub-stitutions that alter the solubilityof the moleculein the red cell. The intraerythrocytic precipitatedmaterial derived from the unstable abnormal Hbis detectable by a supravital stain as dark globu-lar aggregates called Heinz bodies (HBs). Theseintracellular inclusions reduce the life span ofthe red cell and generate a hemolytic process ofvaried severity called congenital Heinz body he-molytic anemia. When a red cell hemolysate ofan affected individual is heated to 508C or treat-ed with 17% isopropanol, a precipitate usuallydevelops. The hemoglobin electrophoresis oftenreveals an abnormal banding pattern. Defini-tive diagnosis requires analysis of either globinstructure or DNA sequence. For more informa-tion on congenital Heinz body hemolytic ane-mia and other hemoglobin variants summa-rized in this section, see Thom et al. (2013).

HEMOGLOBIN VARIANTS WITHALTERED OXYGEN AFFINITY

Over 25 hemoglobin variants have been en-countered in individuals with erythrocytosis.Amino acid substitutions cause an increase inoxygen affinity usually because of impairment



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in the stability of the T or “deoxy” quaternarystructure. Erythrocytosis is usually familial andis the only well-documented clinical finding.Increased oxygen affinity results in a decreasein hemoglobin’s ability to release oxygen duringblood flow in the microcirculation. Unlike in-dividuals with secondary erythrocytosis causedby high altitude exposure or to cardiac or pul-monary hypoxemia, those having hemoglobinwith high oxygen affinity have normal arterialoxygen tension. Therefore, they are not hypox-emic. However, the reduction in unloading ofoxygen to tissues results in cellular hypoxia.Thus, the cells in the kidney that produce eryth-ropoietin sense hypoxia and up-regulate Epogene expression leading to expansion of thered cell mass. About half of the high-affinityvariants can be detected by hemoglobin electro-phoresis. Definitive diagnosis is established bydemonstration of a “shift to the left” in the oxy-hemoglobin binding curve.

Less often, individuals and their relativeswill present with low O2 saturation and cyanosisowing to a hemoglobin variant with a markedreduction in oxygen affinity. The variants thathave been most thoroughly characterized entailamino acid replacements that destabilize the Ror “oxy” quarternary structure. Affected indi-viduals have normal hemoglobin levels and noclinical manifestations other than the duskyskin color.

THE M HEMOGLOBINS

Rare families have attracted medical attentionbecause affected individuals have cyanosis ow-ing to the inheritance of a hemoglobin variantin which an amino acid substitution within theheme pocket results in stabilization of the hemeiron in the oxidized Fe3þ form. As discussed inSchechter (2013), among the most highly con-served amino acids within hemoglobin sub-units are two histidines, one at helix positionF8, which binds to the heme iron and one atE7 in the pocket where oxygen and other ligandsbind to heme. Replacement of either of thesehistidines by tyrosine in either the a-, b-, org-globin subunit results in an alteration in theelectronic environment of the heme iron so that

it is stabilized as Fe3þ. Thus, the M hemoglobinsinclude a58 His! Tyr, a87 His! Tyr, b63His! Tyr, b92 His! Tyr, g63 His! Tyr,and g92 His! Tyr. In addition, b67 Val!Glu results in very similar biochemical featuresand clinical presentation. Affected individualshave cyanosis similar in hue to those with meth-emoglobinemia in which the heme iron in nor-mal hemoglobin has been oxidized to Fe3þ.They have either normal hemoglobin levelsor mild anemia and no clinical manifestationsother than their skin color.

IMPACT OF SUBUNIT ASSEMBLY ONHEMOGLOBIN PHENOTYPE

The composition of the normal and mutant he-moglobins in red cells of heterozygotes providesinsight into the assembly of human hemoglo-bins (Bunn and McDonald 1983). The great ma-jority of b-subunit mutants are synthesized atthe same rate as bA and have normal stability.Therefore, heterozygotes would be expected tohave equal amounts of normal and mutant he-moglobin. However, measurements of the pro-portion of normal and abnormal hemoglo-bins in heterezygotes have revealed unexpectedvariability. Figure 1 shows a comparison of 105stable b-subunit mutants. Positively chargedmutants, such as hemoglobins S, C, D-Los An-geles, and E5, constitute significantly less thanhalf of the total hemoglobin in heterozygotesand are reduced further in the presence of a-thalassemia, because of increased competitionbetween normal and mutantb-subunits for lim-iting amounts of a-subunits. In contrast, manyof the negatively charged mutants are present inamounts exceeding that of HbA. In two hetero-zygotes who had a negatively charged mutant(HbJ-Baltimore or HbJ-Iran) in conjunctionwith a-thalassemia, the proportion of the mu-tant hemoglobin was further increased becausethese bJ-subunits out-compete normal bA-sub-units for limiting amounts of a-subunits.

5Levels of HbE in AE heterozygotes are lower than theothers because the synthesis of bE-globin is impaired owingto a defect in mRNA splicing (see Fucharoen and Weatherall2012 and Thein 2013).



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Analyses of the proportion ofb-subunit mutantin heterozygotes suggest that alterations in sur-facecharge contribute to different ratesofassem-bly of the hemoglobin tetramer. This hypothesisis supported by invitro mixing experiments withnormal and mutant subunits showing that whena-subunits are present in limiting amounts(mimicking a-thalassemia), negatively chargedmutants are formed much more readily than arepositively charged mutants (Mrabet et al. 1986;Adachi et al. 1998).

This electrostatic model of hemoglobin as-sembly has clinical implications. Differencesin rates of assembly explain the higher propor-tion in HbS in sickle C (SC) disease than in sickletrait (AS), a difference that contributes signif-icantly to HbS polymerization and consequentvaso-occlusion in SC patients (Bunn et al. 1982).HbS-Oman is a rare stable mutant with boththe sickle (b6Glu ! Val) substitution andb121Glu! Lys, a substitution that stabilizesthe sickle polymer. Thus, b-subunits of HbS-Oman have a gain of three positive charges.

Accordingly, in red cells of heterozygotes, it ispresent in much less abundance than HbA.However, because of its markedly enhancedpolymerization, heterozygotes with only 20%HbS-Oman and one-gene deletion a-thalas-semia (a-/aa) have hemolytic anemia and signsand symptoms of sickle vaso-occlusion. In con-trast, HbS-Oman heterozygotes with moresevere a-thalassemia (a-/a-) have only 13%HbS-Oman and a normal clinical and hemato-logic phenotype (Nagel et al. 1998).

The impact of surface charge on hemoglo-bin assembly also explains the differences in thelevels of HbA2 (Bunn 1987) and HbF (Adamset al. 1985; Chui et al. 1990) that accompanycertain hematological disorders. For example,a-hemoglobin subunits bind more readily tob than to the positively charged d. Accordingly,HbA2 levels are decreased in the presenceof limiting levels of a-hemoglobin, e.g., a-thalassemia as well as iron deficiency in whichthere is an acquired impairment of a-globinsynthesis.

210

20 S-Oman

E

D-Los AngS

CO-Arab

Korle-Bu

N-Balt

J-Balt

J-Iran

30

40

% o

f tot

al h

emog

lobi

n 50

60

70A B

1 0 –1 –2 αα/αα αα/α– α–/α–

αα/ – –α–/ – –

Figure 1. Impact of surface charge on hemoglobin assembly. (A) Effect of charge on the proportion of abnormalhemoglobin in individuals heterozygous for 105 stable b-globin variants. Each data point represents a meanvalue 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 theproportion of seven positively charged b-subunit variants (blue) and three negatively charged variants (red).(Updated from Bunn and McDonald 1983 and Bunn 1987.)



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POSTTRANSLATIONAL ALTERATIONSIN HEMOGLOBIN STRUCTURE

Awide range of posttranslational modificationsof hemoglobin structure have been identified,some of which result in significant perturba-tions of function.

Nonenzymatic Glycation

The concentration of glucose in the RBC cytosolis nearly that of the plasma. Glucose is in equi-librium between the cyclic hemiacetal structureand the open aldehyde structure. The latter isable to react covalently with amino groups of awide range of proteins. Because this reactionfavors amino groups with relatively low pKas,the most abundant glycated hemoglobin is atthe amino termini of a- and b-globin. In nor-mal red cells, �5% of hemoglobin has a glucoseadduct at the b-amino terminus designatedHbAIc:

HC=O

HCOH

HCOH 1 2β–NH2 +

HOCH

HCOH

Glucose Aldimine Ketoamine

CH2OH

HC=N–β

HCOH

HCOH

HOCH

HCOH

CH2OH

CH2–N+H2–β

C=O

HCOH

HOCH

HCOH

CH2OH

Reaction 1 shows the formation of the Schiffbase or aldimine adduct. This product then re-arranges to the more stable (virtually irrever-sible) ketoamine linkage (reaction 2). HbAIcand other less abundant glucose adducts areformed in a linear manner over the 120-day lifespan of the red cell. Therefore, HbAIc levels arelow in individuals with hemolysis. Because thelevel of HbAIc is an accurate reflection of theaverage concentration of plasma glucose overthe preceding 2 months, it has proven to bevery useful in monitoring therapeutic controlin patients with diabetes.

Amino-Terminal Acetylation

Shortly following translation of most proteins,the amino-terminal (initiator) methionine iscleaved and often the adjacent amino acid is

acetylated at the amino terminus. These post-translational modifications are highly depen-dent on the sequence of neighboring aminoacids. All human globins undergo cleavage ofthe amino-terminal methionine but none un-dergo amino-terminal acetylation except for�20% of g-globin subunits (HbFI). However,rare human hemoglobin variants have beenidentified that have amino acid replacementsin the amino-terminal region that cause bothretention of the initiator methionine and N-acetylation.

Amino-Terminal Carbamylation

Urea in the plasma is in equilibrium with am-monium and cyanate ions. Cyanate is able tobind covalently to the amino termini of a- andb-globin subunits to form adducts. Carbamy-lated hemoglobin is not detectable in normalred cells but can accumulate to measurable lev-els in patients with uremia.

Deamidation

In many proteins, the amino group on certainasparagine residues, particularly when adjacentto a histidine, dissociates leaving aspartic acid.Deamidation does not occur on asparagines innormal globin subunits. However, seven humanvariants have been identified in which partial orcomplete deamidation occurs. Four of these en-tail replacements by asparagine. The deamida-tion does not appear to impact function in anyof these variants.

ACQUIRED ALTERATIONS INHEMOGLOBIN FUNCTION

Methemoglobinemia

Certain drugs and toxins can cause enhancedoxidation of heme iron to form methemo-globin. Above levels of 10%, methemoglobinindividuals appear cyanotic. The extent of he-moglobin oxidation is determined by spectro-photometric analysis of the blood. Methemo-globinemia impairs oxygenation of tissuesbecause the Fe3þ heme is incapable of binding



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to oxygen, and, even more importantly, thepresence of one or two oxidized hemes per tet-ramer stabilizes the R quaternary structure andthereby increases the affinity of the remaininghemes for oxygen. Central nervous and cardio-vascular symptoms and signs begin to appearwhen methemoglobin exceeds 30%. Much low-er levels are seen in individuals with congenit-al methemoglobinemia owing to deficiency incytochrome b5 reductase. Both toxic and con-genital methemoglobinemia can be effectivelytreated by administration of the redox dye meth-ylene blue.

Carboxyhemoglobinemia

Carbon monoxide (CO) binds to heme iron ofhemoglobin with an affinity 210 greater than thatof oxygen. Normal red cells contain a very smallamount of carboxyhemoglobin owing to the re-lease of CO from macrophages following the up-take of senescent red cells and degradation ofheme by heme oxygenase. Higher, albeit nontox-ic, levels of HbCO are present in patients withhemolytic anemias. Inhalation of CO from in-complete combustion of hydrocarbons can resultin toxic carboxyhemoglobinemia. As in methe-moglobinemia, CO poisoning causes morbidityand mortality by ischemia owing to a left shiftin the oxyhemoglobin binding curve, therebyimpairing the unloading of oxygen to cells andtissues. Patients with acute CO poisoning gen-erally develop CNS and cardiovascular symp-toms when levels exceed 20%. Levels of 40%–60% HbCO result in stupor, coma, and death. Allsymptomatic patients should be promptly re-moved from further exposure to CO and treatedwith oxygen. If possible, those with severe toxic-ity should be treated with hyperbaric oxygenadministration or exchange transfusion.

Alterations in Oxygen Affinity

As mentioned above, protons (Hþ) and 2,3-BPG are potent allosteric modulators of oxygenaffinity in normal RBC. Because of their prefer-ential binding to the T or “deoxy” quaternarystructure of hemoglobin, they lower the affinityfor oxygen and therefore shift the oxyhemoglo-

bin binding curve “to the right.” Except underclinical circumstances resulting in either acido-sis or alkalosis, plasma pH and red cell pH aretightly regulated at 7.4 and 7.2, respectively. Incontrast, levels of red cell 2,3-BPG can vary con-siderably in a wide range of clinical settings.Patients with low levels, such as those whohave received large amounts of stored RBCswill have increased oxygen affinity, which couldcompromise tissue oxygenation. Increased lev-els of RBC 2,3-BPG are much more commonlyobserved, primarily in patients with some formof hypoxia. As a result, the “right shift” in theoxyhemoglobin dissociation curve significantlyenhances the unloading of oxygen to tissues andis therefore a major mode of compensation,particularly in patients with severe anemia.

REFERENCES�Reference is also in this collection.

Adachi K, Yamaguchi T, Pang J, Surrey S. 1998. Effects ofincreased anionic charge in the b-globin chain on assem-bly of hemoglobin in vitro. Blood 91: 1438–1445.

Adams JD, Coleman MB, Hayes J, Morrison WT, Stein-berg MH. 1985. Modulation of fetal hemoglobinsynthesis by iron deficiency. N Engl J Med 313: 1402–1405.

Bunn HF. 1987. Subunit assembly of hemoglobin: An im-portant determinant of hematologic phenotype. Blood69: 1–6.

Bunn HF, McDonald MJ. 1983. Electrostatic interactions inthe assembly of haemoglobin. Nature 306: 498–500.

Bunn HF, Noguchi CT, Hofrichter J, Schechter GP,Schechter AN, Eaton WA. 1982. Molecular and cellularpathogenesis of hemoglobin SC disease. Proc Natl AcadSci 79: 7527–7531.

Chui DH, Patterson M, Dowling CE, Kazazian HJ, Ken-dall AG. 1990. Hemoglobin Bart’s disease in an Italianboy. Interaction between a-thalassemia and hereditarypersistence of fetal hemoglobin. N Engl J Med 323:179–182.

� Dailey HA, Meissner PN. 2013. Erythroid heme biosynthesisand 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,Lee C, Rich AM, et al. 2004. Molecular mechanism ofAHSP-mediated stabilization of a-hemoglobin. Cell 119:629–640.

� Fucharoen S, Weatherall DJ. 2012. The hemoglobin E thal-assemias. Cold Spring Harb Perspect Med 2: a011734.

� Higgs DR. 2013. The molecular basis of a-thalassemia.Cold Spring Harb Perspect Med 3: a011718.

Kihm AJ, Kong Y, Hong W, Russell JE, Rouda S, Adachi K,Simon MC, Blobel GA, Weiss MJ. 2002. An abundant



ww

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ersp

ecti

vesi

nm

edic

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erythroid protein that stabilizes freea-haemoglobin. Na-ture 417: 758–763.

� Lettre G. 2012. The search for genetic modifiers of diseaseseverity in the b-hemoglobinopathies. Cold Spring HarbPerspect Med 2: a015032.

Mollan TL, Yu X, Weiss MJ, Olson JS. 2010. The role of a-hemoglobin stabilizing protein in redox chemistry, dena-turation, and hemoglobin assembly. Antioxid Redox Sig-nal 12: 219–231.

Mollan TL, Khandros E, Weiss MJ, Olson JS. 2012. Thekinetics of a-globin binding to a hemoglobin stabilizingprotein (AHSP) indicate preferential stabilization of ahemichrome folding intermediate. J Biol Chem 287:11338–11350.

Mrabet NT, McDonald MJ, Turci S, Sarkar R, Szabo A,Bunn HF. 1986. Electrostatic attraction governs the dimerassembly of human hemoglobin. J Biol Chem 261: 5222–5228.

� Musallam KM, Tahar AT, Rachmilewitz EA. 2012. b-Thalas-semia intermedia: A clinical perspective. Cold SpringHarb Perspect Med 2: a013482.

Nagel RL, Daar S, Romero JR, Suzuka SM, Gravell D,Bouhassira E, Schwartz RS, Fabry M, Krishnamoorthy R.

1998. HbS-Oman heterozygote: A new dominant sicklesyndrome. Blood 92: 4375–4382.

� Nienhuis AW, Nathan DG. 2012. Pathophysiology and clinicalmanifestations of the b-thalassemias. Cold Spring HarbPerspect Med 2: a011726.

� Sankaran VG, Orkin SH. 2013. The switch from fetal toadult hemoglobin. Cold Spring Harb Perspect Med 3:a011643.

� Schechter AN. 2013. Hemoglobin function. Cold SpringHarb Perspect Med 3: a011650.

� Schechter AN, Elion J. 2013. Pathophysiology of sickle celldisease. Cold Spring Harb Perspect Med (to be published).

� Serjeant G. 2013. Natural history of sickle cell disease.Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a011783.

� Thein SL. 2013. Molecular basis of b-thalassemia. ColdSpring Harb Perspect Med doi: 10.1101/cshperspect.a011700.

� Thom CS, Dickson CF, Gell DA, Weiss MJ. 2013. Hemoglo-bin variants: Biochemical properties and clinical corre-lates. Cold Spring Harb Perspect Med doi: 10.1101/cshper-spect.a011858.



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classification of the disorders of hemoglobin

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