Hemoglobin Variants: Biochemical Properties and Clinical Correlatesperspectivesinmedicine.cshlp.org/content/3/3/a011858.full.pdf · Hemoglobin Variants: Biochemical Properties and

Hemoglobin Variants: Biochemical Propertiesand Clinical Correlates

Christopher S. Thom1,2, Claire F. Dickson3, David A. Gell3, and Mitchell J. Weiss2

1Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine,Philadelphia, Pennsylvania 19104

2Hematology Department, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 191043Menzies Research Institute, University of Tasmania, Hobart, Australia

Correspondence: [email protected]

Diseases affecting hemoglobin synthesis and function are extremely common worldwide.More than 1000 naturally occurring human hemoglobin variants with single amino acidsubstitutions throughout the molecule have been discovered, mainly through their clinicaland/or laboratory manifestations. These variants alter hemoglobin structure and biochem-ical properties with physiological effects ranging from insignificant to severe. Studies of thesemutations in patients and in the laboratory have produced a wealth of information on he-moglobin biochemistry and biology with significant implications for hematology practice.More generally, landmark studies of hemoglobin performed over the past 60 years haveestablished important paradigms for the disciplines of structural biology, genetics, biochem-istry, and medicine. Here we review the major classes of hemoglobin variants, emphasizinggeneral concepts and illustrative examples.

Globin gene mutations affecting hemoglobin(Hb), the major blood oxygen (O2) carrier,

are common, affecting an estimated 7% of theworld’s population (Weatherall and Clegg 2001;Kohne 2011). These mutations are broadly sub-divided into those that impair globin proteinsubunit production (thalassemias) and thosethat produce structurally abnormal globin pro-teins (Hb variants). The latter class is mainlycomposed of missense mutations that cause sin-gle amino acid substitutions in the globin pro-tein, resulting in an abnormal, or “variant” Hbtetramer. Less commonly, Hb variants are asso-ciated with deletions, multiple amino acid sub-

stitutions, antitermination mutations, and al-tered posttranslational processing (Table 1).

Naturally occurring Hb mutations cause arange of biochemical abnormalities, some ofwhich produce clinically significant symptoms.The most common and medically important Hbvariants include HbS (Cao and Kan 2012; Lettre2012; Schechter and Elion 2012; Serjeant andRodgers 2012; Williams and Weatherall 2012),HbC (Cao and Kan 2012; Lettre 2012; Schechterand Elion 2012; Serjeant and Rodgers 2012;Williams and Weatherall 2012), HbE (see thesections on Selected Variants that Illustrate Im-portant Aspects of Hemoglobin Biology and

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.a011858

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Table 1. Hb variants that are discussed in this article

NameGlobin site

(fold)Amino acidsubstitution

Molecularmechanism Clinical phenotype

Other biochemicaland laboratory findings

Unstable MutantsBrockton b138 (H16) Ala . Pro Altered secondary

structureHemolytic anemia,

reticulocytosisPhilly b35 (C1) Tyr . Phe Altered a1b1

interface

Hemolytic anemia,

reticulocytosis

Decreased cooperativity,

increased oxygenaffinity

Peterborough b111 (G13) Val . Phe Altered a1b1interface

Hemolytic anemia,reticulocytosis

Decreased oxygenaffinity

Stanmore b111 (G13) Val . Ala Altered a1b1

interface

Hemolytic anemia Decreased oxygen

affinityJ-Guantanamo b128 (H6) Ala . Asp Altered a1b1

interfaceHemolytic anemia Target cells

Khartoum b124 (H2) Pro . Arg Altered a1b1interface

Normal

Prato a1 or a2 31(B12)

Arg . Ser Altered a1b1interface

Anisocytosis,hypochromia

Mildly unstable inisopropanol

Lombard a2 103(G10)

His . Tyr Altered a1b1interface

Anemia

Contaldo a1 or a2103(G10)

His . Arg Altered a1b1interface

Hemolytic anemia

Foggia a2 117(GH5)

Phe . Ser Altered a1b1interface

Microcytosis Rapidly degraded a

chains

Groene Hart a1 119(H2)

Pro . Ser Altered a1b1interface,disrupted AHSPbinding

Hemolytic anemia,microcytosis

Turriff a1 or a2 99

(G6)

Lys . Glu Altered a1b1

interface,disrupted AHSPbinding

Normal Comigrates with HbA1C,

rapidly degraded a

chains

Beziers a1 99 (G6) Lys . Asn Altered a1b1interface,

disrupted AHSPbinding

Normal Comigrates with HbA1C

Hirosaki a2 43(CE1)

Phe . Leu Altered hemepocket

Heinz body hemolyticanemia

Hyperunstable

Terre Haute b106 (G8) Leu . Arg Altered hemepocket

Heinz body hemolyticanemia, dominantinclusion bodythalassemia

Hyperunstable

High Affinity VariantsKempsey b99 (G1) Asp . Asn Unstable T state Erythrocytosis Decreased cooperativityHiroshima b146

(HC3)His . Asp Mutated Bohr

proton donorErythrocytosis Decreased cooperativity,

decreased Bohr effectYork b146

(HC3)

His . Pro Mutated Bohr

proton donor

Erythrocytosis Decreased cooperativity,

decreased Bohr effectCowtown b146

(HC3)His . Leu Mutated Bohr

proton donorErythrocytosis Decreased Bohr effect

Continued

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Table 1. Continued

NameGlobin site




Rahere b82 (EF6) Lys . Thr Altered 2,3DPGbinding site

Erythrocytosis

Providence b82 (EF6) Lys . Asn Altered 2,3DPG

binding site

Erythrocytosis Low oxygen affinity

Helsinki b82 (EF6) Lys . Met Altered 2,3DPGbinding site

Erythrocytosis Decreased Bohr effect

Low Affinity Variants

Kansas b102 (G4) Asn . Thr Unstable R state Cyanosis Decreasedcooperativity

Beth Israel b102 (G4) Asn . Ser Unstable R state Cyanosis Decreasedcooperativity

St. Mande b102 (G4) Asn . Tyr Unstable R state Cyanosis

Methemoglobin VariantsM-Iwate a1 or a2

(F8)His . Tyr Oxidized heme Pseudocyanosis

(Methemoglobinemia)Abnormal visible

spectrum

M-Saskatoon b63 (E7) His . Tyr Oxidized heme Pseudocyanosis(Methemoglobinemia)

Abnormal visiblespectrum

Globin Chain Elongation VariantsConstant

Spring

a2 142

(HC3)

Stop .

Gln

Antitermination

mutant

Microcytosis Decreased mRNA

stabilityCranston b145

(HC3)þCT Frameshift,

elongatedglobin

Hemolytic anemia Increased oxygenaffinity, decreasedcooperativity

Variants with Multiple EffectsHbE b26 (B8) Glu . Lys Unstable, reduced

synthesisMicrocytosis

Bruxelles b41 (C7) or

b42(CD1)

Phe . 0 Altered heme

pocket

Hemolytic anemia,

cyanosis,splenomegaly,reticulocytosis

Heinz bodies, decreased

cooperativity

Warsaw b42 (CD1) Phe . Val Altered hemepocket

Hemolytic anemia,cyanosis

Heinz bodies,decreased

cooperativityHammersmith b42 (CD1) Phe . Ser Altered heme

pocketHemolytic anemia,

cyanosisHeinz bodies,

decreasedcooperativity

Buccuresti-

Louisville

b42 (CD1) Phe . Leu Altered heme

pocket

Hemolytic anemia,

cyanosis

Heinz bodies,

decreasedcooperativity

Zurich b63 (E7) His . Arg Altered hemepocket

Normal, buthypersensitive tooxidative stress

Decreased cooperativity,increased oxygenaffinity, increased CO

affinityJamaica Plain b6 (A3)

and b68(E12)

Glu . Valand Leu. Phe

Altered secondarystructure

Hemolytic anemia,cyanosis,splenomegaly, splenic

sequestration

Unstable, Heinz bodies,sickle cell phenotype,decreased oxygen

affinity

Continued

Hemoglobin Variants

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Variants that Affect Multiple Hemoglobin Func-tions; see also Musallam et al. 2012), and somethalassemias (e.g., “thalassemic hemoglobinop-athies”), all of which are under positive geneticselection because they confer survival advan-tages in areas where malaria is endemic (Weath-erall and Clegg 2001). In addition to these prev-alent mutant proteins, there are also .1000other known naturally occurring Hb variants,which are rare individually but common collec-tively. Most Hb mutants are cataloged on theGlobin Gene database (HbVar, http://globin.bx.psu.edu; Hardison et al. 2002; Giardine etal. 2011). By convention, these variants arenamed after the geographic origin of the affect-ed individual. Although many Hb variants areclinically silent, some produce clinical manifes-tations of varying severity. Analyses of thesevariants, which can be considered to be “exper-iments of nature” (Garrod 1928), have generatedvaluable insights into structure–function rela-tionships within the Hb molecule, with interest-ing and important clinical consequences.

The goal of this work is to provide a succinctconceptual framework for understanding thebiology and clinical implications of Hb vari-ants. We explore major concepts of Hb biologyfollowed by a discussion of selected Hb variantsthat reinforce basic principles. For more exten-

sive reviews of this topic, see Nathan and Oski’sHematology of Infancy and Childhood (Nathanet al. 2009); Hemoglobin: Molecular, Genetic,and Clinical Effects (Bunn and Forget 1986);Disorders of Hemoglobin: Genetics, Pathophysiol-ogy, and Clinical Management (Steinberg et al.2001); and the Globin Gene database (Hardisonet al. 2002; Giardine et al. 2011).

BASIC PRINCIPLES

Hemoglobin Synthesis, Structure, andFunction

Hemoglobin is a heterotetramer composed ofa-like and b-like globin subunits, each boundto a heme prosthetic group. The major functionsof Hb are to transport oxygen (O2) from thelungs to peripheral tissues and carbon dioxide(CO2) from the tissues to the lungs. The kineticsof Hb-O2 binding and release are fine-tuned forthis purpose and adaptable according to devel-opmental ontogeny and metabolic perturba-tions. Moreover, the Hb molecule must limit po-tential problems caused by its associated ironand free O2, reactive molecules capable of inflict-ing damage through the production of reactiveoxygen species. Efforts to understand how Hbstructure imparts these critical functions have

Table 1. Continued

NameGlobin site




Quebec-Chori b87 (F3) Thr . Ile Altered interactionwith HbSpolymer

Normal Promotes HbSpolymerization

D-Ibadan b87 (F3) Thr . Lys Altered interactionwith HbSpolymer

Normal Inhibits HbSpolymerization

Bristol-Alesha b67 (E11) Val . Met Altered hemepocket

Hemolytic anemia,reticulocytosis

Heinz bodies,decreased

cooperativity,decreased Bohr effect,decreased oxygenaffinity

Toms River g67 (E11) Val . Met Altered hemepocket

Anemia, cyanosis Unstable, low oxygenaffinity

For a full listing of hemoglobin variants, see The Globin Gene Server (http://globin.bx.psu.edu; Hardison et al. 2002;

Giardine et al. 2011).

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been ongoing for more than 50 years. Hemoglo-bin was one of the first proteins to be sequenced(Konigsberg et al. 1961; Schroeder et al. 1961;Watson and Kendrew 1961) and the globin geneswere among the earliest to be cloned (Rabbitts1976). In the late 1950s, Perutz and colleaguesdetermined the three-dimensional structure ofHb through X-ray crystallography (Perutz 1960;Perutz et al. 1960). More recent studies refinedthis structure to high resolution (Paoli et al.1996; Park et al. 2006). In addition, O2 and otherligand-binding properties have been measuredin detail for native Hb and many naturallyoccur-ring mutants. All of this work provides a sub-stantial framework for defining the O2 deliveryproperties of Hb, as well as the molecular conse-quences of variant mutations (see, for example,Lehmann 1957; Konigsberg and Lehmann 1965;Shimizu et al. 1965; Perutz and Lehmann 1968;Perutz 1970; Bunn and Forget 1986).

Globin polypeptides are synthesized fromseparate a-like and b-like globin gene clusterslocated on human chromosomes 16 and 11,respectively. Nascent globin chains rapidly in-corporate heme, which stabilizes their nativefolding into Hb subunits composed of sevenor eight a helices named A–H, which fold to-gether into a globular structure (Fig. 1A). Hbsubunits bind O2 and other ligands via the hemeiron buried within an evolutionarily conservedhydrophobic pocket that faces the outside ofHbA tetramers (Fig. 1B). Heme iron is axiallycoordinated to globin proteins by an invarianthistidine residue in helix F8, termed the “prox-imal” histidine (Fig. 1C). The opposite axialposition binds O2, which is stabilized by inter-action with the conserved “distal” histidine inhelix E7 (Fig. 1C). Multiple additional aminoacids within the globin proteins stabilize hemebinding through noncovalent interactions (Fig.1C). Iron must be in its reduced (ferrous, Fe2þ)state for Hb to bind O2. Oxidized or “met” Hb(ferric, Fe3þ) cannot bind O2 and is relativelyunstable, tending to lose hemin and denature.Thus, red blood cells have evolved elaboratemechanisms to maintain Hb in its reduced state(Bunn and Forget 1986; Ganz and Nemeth2012; Schechter 2012). For example, the methe-moglobin reductase system converts methemo-

globin (metHb) to its reduced form. Not sur-prisingly, globin mutations that alter aminoacids within the ligand pocket frequently pro-duce strong functional effects, including desta-bilization, altered affinity for O2, and increasedrates of metHb formation and heme loss (seethe section on Selected Variants that IllustrateImportant Aspects of Hemoglobin Biology).Variants promoting autoxidation are termed“M-Hbs” (see the sections on Selected Variantsthat Illustrate Important Aspects of Hemoglo-bin Biology and Methemoglobin (“M-Type”)Variants). Because Hb gains its distinctive colorfrom the heme group, alterations that affectthe environment of the heme iron, includingchanges in the surrounding amino acids, dif-ferent gas ligands, or redox state, produce char-acteristic changes in visible light absorption.These color changes are used clinically to assessHb-O2 saturation, metHb formation and theeffects of Hb variants.

Within the Hb tetramer, each globin subunitbinds the unlike chain through two distinct in-terfaces, termed a1b1 and a1b2 (Fig. 1D). In-dividual globin subunits form dimers throughthe extremely high affinity a1b1 interaction.Globin chain monomers are relatively unstablecompared with dimers, with a tendency to formintracellular precipitates that damage erythro-cytes, causing hemolytic anemia. Thus, muta-tions that impair thea1b1 interaction can causeerythrotoxicity by favoring the accumulationof monomeric subunits (Fig. 2C). The a1b2 in-teraction, which is lower affinity, mediatestetramerization. Oxygen binding destabilizesthe a1b2 interaction, resulting in a transitionof the quaternary structure from the “T” (tense,low affinity, deoxygenated) to “R” (relaxed, highaffinity, oxygenated) state, which facilitates O2

binding to additional subunits (Fig. 2A). Thisprocess causes cooperative O2 binding, whichis illustrated by the characteristic sigmoidalshape of the Hb-O2 equilibrium curve (Fig.2B). Cooperativity allows maximal O2 releaseover relatively small drops in O2 tension. Muta-tions within the a1b2 interaction region can al-ter the functional properties of Hb, mainly byperturbing O2-binding characteristics (Fig. 2C,cyan spheres; Table 1).

Hemoglobin Variants


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Cooperativity represents a general phenom-enon termed allosteric regulation, in which ef-fector molecules control the properties of en-zymes or other proteins by binding to regionsthat are distinct from the active sites. Several al-losteric regulators, in addition to O2, influencethe properties of Hb. For example, Hþ binds Hbto promote O2 release in a process termed the

Bohr effect (Perutz et al. 1984). In peripheraltissues, abundant CO2 is taken up by red bloodcells and metabolized by carbonic anhydrase tocarbonic acid, resulting in the production of Hþ

and consequent O2 release. In contrast, relativelylow CO2 and high pH in the lung favors O2 bind-ing to Hb. Additionally, CO2 forms carbaminoadducts with the amino termini of botha andb

A

C

D

BD(β)

β1

β1

β

β

α

α

β2

β2

α2

α2

α1

α1

α1β2

interface

α1β1

interface

CB

A

E

F

Phe CD1His E7 His E7

His F8

Phe CD1

Val E11 Val E11

Heme Heme

His F8

H

G

Heme

Figure 1. The structure of Hb. (A) The a (pink) and b (red) Hb subunits have conserved a-helical folds. Helicesare labeled A–H from the amino terminus. The a subunit lacks helix D. (B) The high O2 affinity R statequaternary structure of Hb with O2 (red spheres) bound at all four heme sites (protoporphyrin-IX as yellowsticks, with central iron atom as orange sphere). (C) Stereo (wall-eye) diagram of the heme pocket of b showingthe proximal (F8) and distal (E7) histidines and selected residues in the distal heme pocket that influence ligandbinding and autoxidation. (D) Hb tetramer is assembled from two identical ab dimers (shown in red and grayfor clarity). In the tetramer, each subunit makes contact with the unlike chain through a high affinity dimeriza-tion a1b1 interface and a lower affinity a1b2 dimer–tetramer interface (cyan).

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α2(deoxy) β2(deoxy) β2(oxy)

α1β2interface

α1β1interface

100High O2 affinity

Hb variants

Low O2 affinity Hb variants

O2

Sat

urat

ion

(%)

pH

pH2,3DPGTemp

2,3DPGTemp

H146D (Hiroshima)H146P (York)H146L (Cowtown)

2,3DPGD99N (Kempsey)

K82M (Helsinki)K82N (Providence)

K82T (Rahere)

N102T (Kansas)N102S (Beth Israel)N102Y (St. Mandé)

Y35F (Philly)

F117S (Foggia)

P124R (Khartoum)

R31S (Prato)

A128D (J-Guantanamo)

H103Y (Lombard)

K11E (Turriff)K11N (Beziers)

H103Y (Lombard)H103R (Contaldo)

R31S (Prato)

F117S (Foggia)

AHSP

P119S (Groene Hart)

H103R (Contaldo)

P119S (Groene Hart)V111F (Peterborough)

90

80

70

60

50

40

30

20

10

00 10 20 30 40 50 60 70

pO2(mm Hg)80 90 100

β1

β1

β1

α2(oxy)

α1

α1

α

A

C

B

D

Figure 2. Hb variants with altered subunit interactions. (A) Conversion from the low O2 affinity (deoxy, Tstate) to high O2 affinity(oxy, R state) involves a relative rotation of the a1b1 and a2b2 dimers, with changes in contacts across the a1b2 (and a2b1)interface (cyan). In this cartoon, the a1b1 dimer is held stationary to reveal the relative motion of the a2b2 dimer in going fromthe deoxy (orange) to the oxy (red) states. (B) The sigmoidal shape of the Hb-O2 saturation curve shows allosteric regulation bychanges in pH, temperature, and 2,3DPG. These regulators, as well as Hb variants, influence the shape of the curve. High oxygenaffinity variants, high pH, low 2,3DPG, or low temperature induce a “left shift” in the saturation curve (red line). Conversely, lowoxygen affinity variants, low pH, high 2,3DPG, or high temperature induce a “right shift” (blue line). (C) Hb sequence variants atthe allosteric a1b2 interface (cyan spheres) show an impaired response to O2 binding. Some sequence variants disrupt binding toother allosteric regulators, e.g., substitutions at bK82 (green) disrupt interactions with 2,3DPG that normally stabilize the low O2

affinity Tstate. Mutations that disrupta1b1 (anda2b2) dimerization (blue spheres) increase the concentration of free monomers,which are unstable. (D) Some a mutations that disrupt binding to b may also disturb binding to the chaperone, AHSP. Other avariants, such as Turriff and Beziers (pink sphere) may inhibit only AHSP binding.

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globins in vivo. These interactions produce mi-nor effects on whole blood O2 affinity comparedto the impact of CO2 on pH (Bunn and Forget1986). The compound 2,3-diphosphoglycerate(2,3DPG), formed as a by-product of glycolysisand present at relatively high concentrations inred blood cells, is another important allostericregulator of Hb. 2,3DPG binds and stabilizesT state Hb to facilitate O2 release. Hemoglobinbinding sites for Hþand 2,3DPG have been iden-tified (Perutz et al. 1969, 1984; Perutz 1970; Ar-none 1972). Together, these interactions allowHb to sense metabolic activity and modulateO2 binding accordingly. This environmentalsensory function can be altered by mutationsthat affect the affinity of Hb for its allosteric reg-ulators (Fig. 2C, green sphere; Table 1).

Identification of Hb Variants and TheirClinical Implications

Many Hb variants are readily ascertainedthrough physical examination and/or routinelaboratory testing, which explains why so manyhave been discovered. Uncommonly, some vari-ants are identified through evaluation of ill pa-tients with severe anemia or clinically significantcyanosis. Many amino acid substitutions altersurface charge and are thus detected by electro-phoresis or chromatography, techniques whichare routinely performed on neonates in NorthAmerica and Europe. Interestingly, a few Hb var-iants migrate similarly to HbA1c, a glycosylatedform of Hb that reflects long-term control ofblood glucose levels in diabetic patients (Table1) (reviewed in Little and Roberts 2009). In thisway, specific Hb amino acid substitutions canartificially elevate HbA1c measurement and in-terfere with diabetic management. Other Hbvariants are clinically benign but produce obvi-ous changes in skin color. For example, muta-tions that increase Hb-O2 affinity typically stim-ulate erythropoietic drive by inhibiting O2 tissuedelivery, causing erythrocytosis associated witha ruddy complexion (Nathan et al. 2009). Mu-tations that reduce O2 affinity produce a bluishhue to the skin (cyanosis) caused by abnormal-ly high levels of deoxyHb. Mutations that favoroxidation of Hb iron to the met form (M-Hbs),

also cause blue-tinged skin, whereas the blooditself appears brown. Studies of one family withcongenital cyanosis led Horlein and Weberto describe the first known hemoglobinopathy,caused by the variant Hb-M Saskatoon (Horleinand Weber 1948). The M-Hb variant Iwate,which causes “black blood” or “hereditary nigre-mia,” was first described more than 200 years agoin Japan (Shibata et al. 1960).

It is important to note that many Hb variantsaffecting skin color are not clinically damag-ing beyond theircosmeticeffects. However, theseconditions can mimic more life-threateningproblems such as cardiopulmonary and myelo-proliferative disorders, which must be excluded.Thus,patientswith cyanoticorpolycythemic Hbvariants may mistakenly undergo unnecessaryand potentially dangerous medical procedures.Historical examples include cyanotic patientsundergoing surgery or catheterization for pre-sumed heart defects and polycythemic patientsreceiving radioactive 32P for presumed polycy-themia vera (Steinberg et al. 2001; Nathan et al.2009). As stated, “the primary reason for estab-lishing the diagnosis [of M Hbs] is to preventiatrogenic misadventures that might arise underthe mistaken impression that the patient has acardiac or pulmonary disorder” (Bunn and For-get 1986). Most Hb variants with altered O2 af-finity are relatively simple to diagnose throughhistory, physical examination, and laboratorytesting (Wajcman et al. 2001; Wajcman and Mo-radkhani 2011).

Globin Gene Synthesis Is DevelopmentallyRegulated

Developmental regulation of the a-like and b-like globin gene families is of great medicalsignificance (Sankaran and Orkin 2012). Hemo-globin F (a2g2) is the most highly expressedform during late fetal gestation. After birth, ex-pression gradually switches to HbA (a2b2) overseveral months. Thus, symptomatic mutationsaffecting a or g globins are present prenatally orat birth, whereas the manifestations of b-glo-bin mutations are typically delayed until a fewmonths after birth. Interestingly, g-globin genemutations that are apparent at birth, most

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typically reflected by cyanosis or hemolytic ane-mia (e.g., Hb-F Toms River, sections on SelectedVariants that Illustrate Important Aspects ofHemoglobin Biology and Variants that AffectMultiple Hemoglobin Functions, and Table 1),fade over a few weeks as Hb production switchesfrom F (a2g2) to A (a2b2).

Laboratory Testing for Hb Variants

In many countries, routine testing of all new-borns is performed to identify common hemo-globinopathies such as some thalassemias andHbS. Isoelectric focusing or high-performanceliquid chromatography (HPLC), the most com-monly used tests, identify most structurally ab-normal Hbs (Wajcman et al. 2001). In this way,many benign Hb variants are discovered inci-dentally. Clinical indications for laboratory test-ing to investigate potential Hb variants are listedin Table 2.

Specific laboratory tests to investigate Hbvariants include:

a. Physical methods of Hb separation. Theseinclude electrophoretic and chromato-graphic techniques that examine the physi-cal properties ofa1b1 dimers or individualglobin subunits. Specific standards, suchas HbA, HbS, HbC, HbF, and HbA2, aretypically examined as controls. Hb variantsmay show altered migration in these assays.Historically, cellulose acetate and citrateagar electrophoresis were most commonlyused to detect variant Hbs. Current clinical

testing more typically uses isoelectric focus-ing and HPLC, which are more sensitive.

b. Hb-O2 binding curve. This test, performedon whole red blood cells or hemolysate, in-dicates the percent (%) oxygenated Hb at agiven O2 partial pressure (Fig. 2B). Hemo-globin variants with an abnormally high O2

affinity (see sections on Selected Variantsthat Illustrate Important Aspects of Hemo-globin Biology and High Oxygen AffinityVariants) will become saturated at lowerO2 pressures producing a “left-shifted” O2

equilibrium curve, whereas mutations thatreduce O2 affinity (see sections on SelectedVariants that Illustrate Important Aspectsof Hemoglobin Biology and Low OxygenAffinity Variants) will cause the opposite“right shift.” Determination of Hb-O2 af-finity responses to allosteric regulators, par-ticularly 2,3DPG or Hþ (pH changes),can provide insight into the structural caus-es of the observed phenotypes. Unfortu-nately, few clinical laboratories currently of-fer this assay.

c. Visible wavelength spectroscopy. Hemoglo-bin variants with amino acid substitutionsin the heme pocket affect visible light ab-sorbance. For example, M-type Hbs showcharacteristic spectra that can distinguishthem from methemoglobinemia caused byan enzyme deficiency in the metHb reduc-tase system (Bunn and Forget 1986; Daileyand Meissner 2012; Ganz and Nemeth2012; Schechter 2012). Pulse oximetry is anoninvasive spectrophotometric test thatmeasures absorbance ratios at specificwavelengths for oxygenated (660 nm) anddeoxygenated (940 nm) blood (Tremperand Barker 1989). This can produce confus-ing and sometimes misleading results in pa-tients with variant Hbs that show uniquelight absorbance properties (Verhovsek etal. 2010). In these cases, analysis of arteri-al blood O2 concentration may be requiredto rule out hypoxia. Analyzing these variantHbs using light absorbance throughout thefull visible wavelength spectrum can pro-vide useful diagnostic information.

Table 2. Clinical indications for laboratory testing todiagnose Hb variants

Indications for hemoglobin testing

Routine newborn testing for commonhemoglobinopathies (i.e., HbS, HbC,thalassemias)

Cyanosis with adequate arterial oxygenation and noapparent cardiopulmonary disease

Erythrocytosis with normal or elevatederythropoietin levels

Unexplained hemolytic anemiaUnexplained thalassemia phenotypeFamily history consistent with an Hb variant

Hemoglobin Variants


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d. Hemoglobin stability testing. Typically, Hbstability is impaired in variants that areassociated with hemolytic anemia. Hemo-globin stability tests measure the propensityfor Hb to denature on exposure to variousstresses including heat (Carrell and Kay1972), isopropanol (Bender et al. 1981),mechanical agitation (Asakura et al. 1975),and zinc acetate (Roth et al. 1976). TheHeinz body test uses supravital stains,such as methylene blue or crystal violet,to detect aggregated globins within ery-throcytes (Eisinger et al. 1985).

e. Specialized testing. Mass spectrometry anal-ysis of patient hemolysate and DNA se-quencing of globin genes are specializedconfirmatory tests to identify amino acidand nucleotide alterations associated withsuspected Hb variants (Wajcman et al.2001; Wajcman and Moradkhani 2011).DNA sequencing may readily elucidate thepresence of an Hb variant. However, addi-tional biochemical and structural studies,including those discussed in this section,are required to determine how the variantaffects Hb function. Crystallographic anal-ysis is the highest resolution approach todetermine the effects of globin amino acidsubstitutions on molecular structure. Crys-tallography has been used historically as aresearch tool to assess the effects of someinteresting Hb variants (see, for example,Pulsinelli et al. 1973; Perutz et al. 1984).

SELECTED VARIANTS THAT ILLUSTRATEIMPORTANT ASPECTS OF HEMOGLOBINBIOLOGY

Unstable Variants

Unstable variants frequently cause congenitalHeinz body hemolytic anemia detected by labo-ratory screening and clinical symptoms (see sec-tions on Basic Principles and Laboratory Testingfor Hb Variants and Table 2). Mutations thatalter any step in globin processing, includingsubunit folding, heme interaction, dimeriza-tion, or tetramerization, can destabilize Hb.Bunn and Forget note five general mechanisms

that destabilize Hbs: amino acid substitutionswithin the heme pocket, disruption of secondarystructure, substitution in the hydrophobic inte-rior of the subunit, amino acid deletions, andelongation of the subunit (Bunn and Forget1986).

More than 75% of Hb is a helical (Perutzet al. 1960; Park et al. 2006). This structure isparticularly susceptible to disruptions by pro-line substitutions (Levitt 1981). For example,in Hb Brockton (b138 [H16] Ala . Pro) thesubstituted proline disrupts intermolecular hy-drogen bonding between b138Ala and b134Valin helix H (Russu and Ho 1986; Moo-Penn et al.1988). This produces an unstable variant with apropensity to precipitate and aggregate, there-by damaging erythrocytes and predisposing tohemolysis. Hb Brockton does not show alteredO2 binding affinity or electrophoretic mobilityshifts. This variant was identified by HPLC anal-ysis of patient globin chains and its altered X-raycrystallography diffraction pattern shows localdisruption of helix H (Moo-Penn et al. 1988).

Mutations at the a1b1 interface can causehemolytic anemia by inhibiting heterodimerformation, favoring the accumulation of freeglobin subunits, which themselves are unstable,particularlya chains (Fig. 2C, blue spheres). Ex-amples include Hb Philly (b35 [C1] Tyr . Phe)(Rieder et al. 1969), Hb Peterborough (b111[G13] Val . Phe) (King et al. 1972), Hb Stan-more (b111 [G13] Val . Ala) (Como et al.1991), and Hb J-Guantanamo (b128 [H6] Ala. Asp) (Martınez et al. 1977). Hb Khartoum(b124 [H2] Pro . Arg) contains a substitutionat the a1b1 interface that is mildly destabilizingin vitro, but does not cause clinical symptoms(Clegg et al. 1969; Argos et al. 1979).

Interestingly, some a-globin (HBA) genemutations affecting the a1b1 interface mayalso destabilize free a chains by inhibiting theirbinding to a-hemoglobin stabilizing protein(AHSP), an erythroid molecular chaperonethat facilitates Hb assembly (Fig. 2D, bluespheres) (reviewed in Weiss et al. 2005; Faveroand Costa 2011). These a-globin variants in-clude Hbs Prato (a1 or a2 31 [B12] Arg .

Ser) (Marinucci et al. 1979), Lombard (a2 103[G10] His . Tyr) (Hoyer et al. 2002), Contaldo

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(a1 ora2 103 [G10] His . Arg) (Sciarratta et al.1984), Foggia (a2 117 [GH5] Phe . Ser) (La-cerra et al. 2008), Groene Hart (a1 119 [H2] Pro. Ser) (Harteveld et al. 2002; Vasseur-Godbil-lon et al. 2006; Giordano et al. 2007; Vasseuret al.2009), and others (Wajcman et al. 2008; Yu et al.2009). Naturally occurring a-globin variantswith amino acid substitutions at position 99 in-cluding Hb Turriff (a1 ora2 99 [G6] Lys . Glu)(Langdown et al. 1992) and Hb Beziers (a1 99[G6] Lys . Asn) (Lacan et al. 2004) bind b glo-bin normally but show impaired interactionwith AHSP and may be mildly destabilizing(Fig. 2D, violet spheres) (see also Yu et al. 2009;Khandros et al. 2012; Mollan et al. 2012).Antitermination mutations can also destabilizea globin in part by impairing its binding toAHSP (Turbpaiboon et al. 2006).

Hyperunstable Hb variants precipitateshortly after synthesis and are not incorporatedinto Hb tetramers. These ephemeral proteinsare difficult to isolate. In this case, electropho-resis may be falsely negative due to the rapidturnover of these variants, making DNA se-quencing a critical diagnostic test. Affected pa-tients show a dominantly inherited “inclusionbody thalassemia” resulting from both the pre-cipitated variant globin and consequent chainimbalance with accumulation of the unaffectedglobin, which is unstable in its free form (Sta-matoyannopoulos et al. 1974). Patient erythro-cytes typically display abnormal morphologywith microcytosis, hypochromia, moderate tosevere anisopoikilocytosis, basophilic stippling,and inclusions that may be become particularlyprominent following splenectomy (Steinberget al. 2001). Weatherall, Thein, and colleaguescharacterized several hyperunstable mutationsin exon 3 of the b-globin gene (Thein et al.1990). All of the mutations were frameshiftsor nonsense codons that produced relativelylong (.120 amino acid) proteins with car-boxy-terminal truncations. The investigatorsproposed that truncated globins causing dom-inantly inherited thalassemia are long enoughto bind heme posttranslationally, which ren-dered them relatively resistant to proteolyticdegradation, allowing for subsequent precip-itation of heme-containing aggregates detected

as Heinz bodies. Missense mutations also causehyperunstable Hb variants. Hb Hirosaki (a2 43[CE1] Phe . Leu) was discovered in a familywith hemolytic anemia (Ohba et al. 1975; Tana-ka et al. 2005). After several tests failed to iden-tify a soluble variant Hb within erythrocytes,DNA sequencing was used to characterize themutation. Hb Terre Haute (b106 [G8] Leu .

Arg) is another hyperunstable variant associatedwith a severe Heinz body hemolytic anemia andglobin chain imbalance (Coleman et al. 1991).In initial studies of patient erythroid cells, per-formed in 1979, abnormal Hb tetramers werenot detected and peptide mapping of radiola-beled nascent globins identified a b112 (G14)Cys . Arg substitution, originally termed HbIndianapolis (Adams et al. 1979). However, theb112 Cys . Arg mutation was subsequentlyidentified in unrelated individuals with muchless severe disease. In 1991, reevaluation of theoriginal pedigree by DNA analysis discovered ab106 Leu . Arg mutation, which was renamedHb Terre Haute (Coleman et al. 1991). Mostlikely, incomplete tryptic cleavage of the abnor-malb-globin peptide in the earlier studies led tomisidentification of the causative mutation.This work reflects the interesting historical pointthat many Hb variants were identified throughlaborious and technically challenging proteinstudies performed some time ago, before DNAsequence analysis of patient globin genes wasfeasible. Reevaluation of these mutationsthrough genetic testing has yielded some inter-esting surprises (see also the discussion on HbBristol-Alesha and the section on Variants thatAffect Multiple Hemoglobin Functions).

High Oxygen Affinity Variants

Hemoglobin variants with increased O2 affinitycause erythrocytosis by stimulating erythro-poietic drive (see Hebbel et al. 1978 for a de-scription of the associated physiology). Thiscommonly results from amino acid substitu-tions that stabilize the R (high O2 affinity) staterelative to the T (low O2 affinity) state and/orinhibit responses to environmental allostericregulators that stimulate O2 release, includingHþ (Bohr effect) or 2,3DPG (see the sections on

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Basic Principles and Hemoglobin Synthesis,Structure, and Function). Because T to R statetransitions are mediated largely through a1b2interactions, high affinity variants frequently re-sult from substitutions that alter this interface(Fig. 2C, cyan spheres). For example, the aminoacid replacement in Hb Kempsey (b99 [G1] Asp. Asn) perturbs a1b2 interactions by prevent-ing the formation of a hydrogen bond betweenb99 Asp and a42 Tyr, which normally stabilizesthe deoxygenated low O2 affinity T state (Fig. 3)(Reed et al. 1968; Lindstrom et al. 1973; Bunnet al. 1974). This structural change shifts quater-naryequilibrium toward the oxygenated R form,which impairs O2 release to peripheral tissuesand thus increases erythropoietic drive. The car-boxyl termini of globin chains are also involv-ed in a1b2 interactions that stabilize the lowO2 affinity T state and numerous substitutions

within these regions cause high O2 affinity vari-ants. In addition, b146 His at the carboxyl ter-minus contributes significantly to the Bohr ef-fect by forming a salt bridge with b94 Asp(Perutz et al. 1984). This interaction is disruptedin several high O2 affinity variants with b146substitutions: Hb Hiroshima (b146 [HC3] His. Asp) (Hamilton et al. 1969; Perutz et al. 1971;Imai etal.1972;Olsonetal.1972),HbYork(b146[HC3] His . Pro) (Bare et al. 1976), and HbCowtown (b146 [HC3] His . Leu) (Fig. 2C)(Schneider et al. 1979; Perutz et al. 1984). Thesevariants show reduced Bohr effect with im-paired release of O2 under acidic conditions.

Several high affinity Hb variants are causedby substitutions that inhibit interaction with2,3DPG, which normally binds globin chainsto stimulate O2 release (Fig. 2C). For example,Hb Rahere (b82 [EF6] Lys . Thr) replaces an

α2

β2

β2 β2

β1

α1

α1 α1

αN102T

(Kempsey)

A

B

αD99N

(Kansas)

Y42 Y42W37W37

N97 N97

D99 D99D94

D94

N102 N102

Deoxy-Hb (T state) Oxy-Hb (R state)

Figure 3. Hb variants that affect allosteric regulation. (A) The a2 and b2 subunits indicated on the right side ofthe figure show an overlay of the R-state (red/pink) and T-state (orange/light orange) quaternary structures ofHb. The allosteric a1b2 interface is boxed. (B) Detail of the a1b2 interface in the deoxy T state (b2 chain inorange, PDB 2DN2) and oxy R state (b2 chain in red, PDB 2DN1) showing selected H-bonding interactions.Note that the two H-bonding networks use nonoverlapping sets of side chains, hence mutations in these residuesaffect only one state. Mutation of Asp99 to Asn (Hb Kempsey) compromises electrostatic interactions in thedeoxygenated state, thereby favoring the R state and causing impaired O2 release. Mutation of Asn102 to Thr(Hb Kansas) abrogates interactions with Asp94, favoring the T state and O2 binding inhibition.

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invariant lysine in the 2,3DPG binding site ofb globin, thereby reducing the affinity for thisallosteric regulator (Lorkin et al. 1975; Sugi-hara et al. 1985). Consequently, Hb Rahereshows blunted O2 release in response to added2,3DPG in vitro. In vivo, O2 release in peripheraltissues is inhibited, resulting in elevated bloodHb levels. Similarly, Hb Providence (b82 [EF6]Lys . Asn) (Bonaventura et al. 1976; Moo-Penn et al. 1976) and Hb Helsinki (b82 [EF6]Lys . Met) (Ikkala et al. 1976; Charache et al.1977) are high O2 variants caused by differentamino acid substitutions at the 2,3DPG bindingsite on b82.

Low Oxygen Affinity Variants

Low O2 affinity Hb variants typically presentwith cyanosis. In general, these variants arecaused by globin amino acid substitutions thattip the quaternary equilibrium of Hb tetramersfrom the high affinity oxygenated R state to thelow affinity deoxygenated T state; more or lessthe opposite of what occurs for high O2 affinityvariants (see the sections on Selected Variantsthat Illustrate Important Aspects of Hemoglo-bin Biology and High Oxygen Affinity Variants).This does not inhibit Hb-O2 release in tis-sue capillaries, but rather, interferes with Hb-O2 uptake if the P50 has increased to �50 mmHg. Paradoxically, low O2 affinity Hb variantscan be associated with mild anemia thought tobe caused by increased O2 tissue delivery withreduced erythropoietic drive (Stamatoyanno-poulos et al. 1969). In addition, some low O2

affinity mutants are unstable and therefore as-sociated with not only cyanosis but also Heinzbody hemolytic anemia (see the sections on Se-lected Variants that Illustrate Important Aspectsof Hemoglobin Biology and Unstable Variants).

Several low affinity variants involve replace-ments at the a1b2 interface, which plays animportant role in Hb cooperativity. Hb Kansas(b102 [G4] Asn . Thr) is a well-studied low O2

affinity variant (Fig. 3B) (Reissmann et al. 1961;Bonaventura and Riggs 1968; Gibson et al. 1973;Riggs and Gibson 1973). Affected individualsare markedly cyanotic, although clinically well.Replacement of Asn102 at the a1b2 interface

inhibits the formation of a hydrogen bondwith Asp94 that normally stabilizes the oxygen-ated R structure. A similar mechanism causeslow O2 affinity in two other Hb variants throughdifferent substitutions of the same amino acidresidue (B102 [G4] Asn) in Hb Beth Israel (b102[G4] Asn . Ser) (Nagel et al. 1976) and HbSt. Mande (b102 [G4] Asn . Tyr) (Arous etal. 1981; Poyart et al. 1990).

Methemoglobin (“M-Type”) Variants

Hemoglobin iron must be in its reduced (Fe2þ,ferrous) state to bind O2. Moreover, oxidized(Fe3þ, ferric, met) Hb is intrinsically unstablewith a tendency to release heme. Hemoglobinreduction is maintained through intrinsic fea-tures of the Hb protein and extrinsic antioxi-dant pathways within red blood cells (see thesections on Basic Principles and HemoglobinSynthesis, Structure, and Function). Exposureto oxidant drugs or toxins, genetic alterationsin erythroid metHb reductase enzyme systems(Ganz and Nemeth 2012; Schechter 2012), orglobin chain variants can predispose to met-hemoglobinemia. These disorders present as“pseudocyanosis,” (i.e., low Hb-O2 saturation),despite adequate arterial oxygenation. Detailedin vitro analyses of the red cell and isolated Hbsamples can usually distinguish wild-typemetHb resulting from toxins or defective reduc-tase systems and M-type Hb variants that arepredisposed to spontaneous oxidation (Bunnand Forget 1986; Steinberg et al. 2001; Nathanet al. 2009). For example, various M-Hbs showcharacteristic visible absorbance spectra.

Globin variants associated with metHb for-mation are typically caused by amino acid sub-stitutions within the heme pocket. For example,four different M-Hbs occur when tyrosine re-places the a- or b-globin proximal or distal his-tidine residues that interact with heme (Fig. 1C)(reviewed in Adachi et al. 2011). In Hb M-Iwate(a1 or a2 87 [F8] His . Tyr), the proximalhistidine is replaced by tyrosine (Fig. 4A), whichdeprotonates and coordinates to the heme iron(Fig. 4B) (Konigsberg and Lehmann 1965; Shi-mizu et al. 1965). Ferric heme, bound throughthe native His F8, is readily reduced by metHb

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reductase (Fig. 4C). Tyrosine (F8) coordinationstabilizes the oxidized ferric state and decreasesreactivity with metHb reductases. This interac-tion also distorts the position of heme and helixF within the altered a subunits. In native Hb,movement of the proximal His F8 and F helixaway from the heme group stabilizes the deox-ygenated T state and reduces the O2 affinity ofthe native b subunit partners. Hence, substitu-tion of the normal His F8 side chain for a longerTyr F8 (Fig. 4A) also stabilizes the deoxygenatedT state and reduces O2 affinity of the native b

subunit in Hb M-Iwate (Nagai et al. 2000; Jinet al. 2004). These biochemical and structuralalterations underlie the lack of cooperativityand severe cyanosis in patients with Hb M-Iwate, in which metHb levels can exceed 20%(normal ,2%) (Ameri et al. 1999). In contrast,Hb M-Saskatoon (b63 [E7] His . Tyr) re-places the distal His with Tyr (Fig. 4D) (Horleinand Weber 1948; Hayashi et al. 1966). In this

variant, the protonated form of mutant Tyr canbind ferric heme iron to generate a hexacoordi-nate structure that is relatively easily reduced bycellular metHb reductases (Fig. 4E). Conse-quently, patients with Hb M-Saskatoon havelower levels of circulating oxidized Hb com-pared with those with Hb M-Iwate. Compara-tive studies of these patients and their variantM-Hbs have contributed greatly to understand-ing the biochemical properties of the heme iron,including its interactions with various ligandsand nearby amino acids such as the proximaland distal histidines.

Globin Chain Elongation Mutants

Antitermination and frameshift mutations thatadd irrelevant amino acids to the carboxyl ter-minus of globin proteins produce interestingvariants that can damage erythrocytes (Nathanet al. 2009). The most clinically significant

Iwate (α F8 Tyr)

(β E7 Tyr)Saskatoon

Tyr F8

E

BA C

E7H+

HO–

NN

NN

NN

F8

Normal

HH2O

Fe3+

E7

F8

Fe3+

Fe3+ Fe3+

O

N N metHbreductase

system

metHbreductase

system

N

N

Fe2+

N

N

O

Fe2+

OH

Tyr E7

D

HN N

HN N

Figure 4. Examples of M-type hemoglobins. (A) The heme pocket of wild-type Mb (gray) and the F8 His . Tyrmutation (orange, PDB 1HRM), which serves as a model for Hb M-Iwate. Effects on the protein fold are toincrease the distance from the heme to the F helix, recapitulating features of the deoxy-a (Tstate) structure. (B)In Hb M-Iwate,a 87 Tyr F8 is deprotonated and favors Fe3þ oxidation state, resulting in rapid autoxidation. Thisferric form is not reduced by metHb reductase. (C) With the normal His F8 present, ferric heme is readilyreduced. (D) Substituting the distal His E7 side chain in Mb for a larger Tyr E7 (orange, PDB 1MGN), as alsooccurs in Hb Saskatoon, brings the Tyr hydroxyl group within binding distance of the iron, forming a hex-acoordinate iron site. (E) In Hb Saskatoon the hexacoordinate ferric iron in the effected b chains can be reducedby metHb reductase, possibly owing to a transient protonation of Tyr E7. Note that Mb is used as a model for Hbsubunits in A and D, whereas B and E are based on Hb spectroscopy.

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example is Hb Constant Spring (a2 142 [HC3]Stop . Gln), caused by an antitermination mu-tation at the a2 stop codon (Clegg et al. 1971;Efremov et al. 1971; Milner et al. 1971; Cleggand Weatherall 1974). This elongates the pro-tein by 31 amino acids, generating an unstableprotein that is relatively underrepresented inhemolysates, but can be detected by physicalmethods (see sections on Basic Principles, Lab-oratory Testing for Hb Variants, and Physicalmethods of Hb separation). In addition, HbConstant Spring mRNA is rapidly degraded indeveloping erythrocytes, owing to ribosomalentry into the 30UTR, causing displacement ofRNA-bound stabilizing proteins with a resul-tant thalassemia syndrome (Hunt et al. 1982;Derry et al. 1984; Weiss and Liebhaber 1994;Morales et al. 1997).

Hb Constant Spring contributes toa-thalas-semia syndromes, particularly when combinedwith two a-globin deletional alleles (–/aCSa),which produces a severe form of HbH disease(Viprakasit and Tanphaichitr 2002). IsolatedHb Constant Spring, in its heterozygous (aa/aCSa) or homozygous (aCSa/aCSa) forms, re-sults in more severe anemia than occurs whenthe same a alleles are deleted (aa/2a) or(2a/2a) (Schrier et al. 1997). This is due tothe cytotoxic effects of the unstable ConstantSpring protein. Although most common inSoutheast Asia, Hb Constant Spring is increas-ingly identified in other geographic regions,largely through global migration (Lal et al.2011). In fact, it was first discovered in a Chinesefamily living in Constant Spring, Jamaica.

One example of ab-globin chain elongationmutant is Hb Cranston (b 145 [HC3] þCT)(Bunn et al. 1975). This mutation introduces aframeshift at the normal stop codon to generatea b chain that is extended by 11 amino acids.This results in an unstable Hb tetramer withhigh O2 affinity and diminished cooperativity(McDonald et al. 1980; Shaeffer et al. 1980). Af-fected patients show compensated hemolyticanemia with the variants accounting for 30%of total Hb in the hemolysate. Interestingly, thestructure of Hb Cranston was investigatedsimultaneously with studies to determine theb-globin mRNA 30 untranslated sequence (For-

get et al. 1975). Cross-comparison of the proteinand mRNA sequencing data allowed Bunn, For-get, and colleagues to more rapidly define nor-mal b-globin gene structures and ascertain thatthe Hb Cranston mutation likely arose by non-homologous crossover of two normal b-globingenes.

Variants that Affect Multiple HemoglobinFunctions

Not surprisingly, amino acid substitutions with-in critical regions of globin proteins can pro-duce multiple effects. For example, HbE (b26[B8] Glu . Lys), a common variant in South-east Asia, contains an amino acid substitutionthat renders b chains mildly unstable in vitrowith minimal clinical significance (Frischerand Bowman 1975; Huisman 1997; Rees et al.1998; see also Musallam et al. 2012). However,this mutation also creates an alternate splice sitein the b-globin mRNA, leading to reduced syn-thesis of productive transcripts with resultantthalassemia (Orkin et al. 1982). HbE is particu-larly deleterious when coinherited with a moresevereb-thalassemic allele, which happens com-monly in Southeast Asia.

Mutations that alter the heme pocket com-monly produce multiple biochemical effects.For example, deletion or substitution of theconserved Phe residue at the CD1 helical regionin the heme pocket markedly destabilizes theaffected globin and also alters its O2 affinity.Thus, Hb Bruxelles (b42 [CD1] Phe . 0)(Blouquit et al. 1989; Griffon et al. 1996), HbWarsaw (b42 [CD1] Phe . Val) (Honig et al.1990), Hb Hammersmith (b42 [CD1] Phe .

Ser) (Dacie et al. 1967), and Hb Buccuresti-Lou-isville (b42 [CD1] Phe . Leu) (Bratu et al.1971; Keeling et al. 1971) cause both congenitalHeinz body hemolytic anemia and cyanosis.These combined effects arise from severely re-duced cooperativity, rapid rates of autoxidationand hemin loss, and unfolding of these unstableglobin variants (Griffon et al. 1996).

Another interesting heme pocket variant isHb Zurich (b63 [E7] His . Arg) in which thedistal His is replaced by Arg (Huisman et al.1961; Bachmann and Martihr 1962; Frick et al.

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1962; Tucker et al. 1978; Phillips et al. 1981;Springer et al. 1989). The large, highly polarvariant His side chain swings out of the distalheme pocket, and the positively charged guani-dino group forms a salt bridge with a hemepropionate (Fig. 5A). This results in an enlargedligand-binding pocket, destabilizing O2 bindingand causing iron autoxidation via exposure towater. Affected individuals show increased sen-sitivity to oxidant agents, including sulfa drugs,which more easily enter the expanded hemepocket. Loss of the distal histidine markedlydecreases O2 affinity but has little effect on car-

bon monoxide (CO) binding. As a result, indi-viduals with Hb Zurich tend to have supranor-mal levels of CO-Hb, which ironically protectsthe heme iron from oxidation and the globinfrom denaturation. Affected subjects who arecigarette smokers accumulate even higher levelsof CO-Hb, which tends to protect against hemo-lysis. Thus, “the pathology of a mutant protein isameliorated by a normally toxic pollutant”(Bunn and Forget 1986).

Two other recently identified Hb variantsillustrate how multiple biochemical defectsproduce unique phenotypes. Hb Jamaica Plain

Phe CD4

Phe CD1

Phe CD1

His F8

A

B

His F8

Arg E7

Phe CD4

Phe CD1

Phe CD1

His F8

His F8

Arg E7His E7His E7

His E7 His E7

Trp E11 Trp E11Thr E11 Thr E11Val E11 Val E11

Figure 5. Hb variants with amino acid changes in the heme pocket. (A) Stereo diagram of a model of the deoxyHb Zurich b heme pocket (blue) overlaid with the wild-type b heme pocket (black, PDB 2DN2). This model ofHb Zurich is based on the structures solved by Phillips et al. (1981) and Tucker et al. (1978). It was generated withthe macromolecular modeling program Coot (Emsley et al. 2010) by mutating the distal His of deoxy-b (PDB2DN2) to Arg. The distal Arg E7 (red) is oriented toward the CD corner, disturbing the position of Phe CD1 andPhe CD4 (orange). The heme pocket entrance is much wider allowing increased access to ligands. However,unlike normal His E7, mutant Arg is unable to stabilize bound O2 via hydrogen bonding. (B) Stereo diagramshowing the structural changes associated with substitution of b Val E11. The wild-type structure carrying thebranched hydrophobic side chain Val (black bonds, PDB 2DN2) is overlaid with structures carrying the largestaromatic side chain Trp E11 (orange, PDB 101K) or a polar side chain Thr E11 (green PDB, 1HDB). It is clearthat no major backbone or side chain repacking occurs. Thus, mutations in this position are likely to havespecific effects in changing the volume of the heme pocket accessible to solvent or diatomic ligands and theelectrostatic properties of the pocket. These changes will manifest as differences in O2 binding, ligand selectivity,autoxidation, and heme loss.

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(b6 [A3] Glu . Val and b68 [E12] Leu . Phe)contains two defects in the same b chain, a b6Glu to Val substitution that causes sickle cellanemia (Serjeant and Rodgers 2012) and a b68amino acid substitution that reduces O2 affini-ty, probably by destabilizing the oxygenatedconformation through steric effects introducedinto helix E (Geva et al. 2004). The affected pa-tient, who was heterozygous for the mutant al-lele, showed symptoms of sickle cell anemia thatwere precipitated by infection or airplane travel.Thus, an amino acid substitution that reducesO2 affinity exacerbates the effects of a sicklingmutation in the same globin chain.

Several other Hb variants modulate the se-verity of sickle cell anemia (see also Cao and Kan2012; Schechter and Elion 2012; Serjeant andRodgers 2012). For example, g globin inhibitspolymerization of HbS (Nagel et al. 1979). Thiseffect is attributable to differences in severalamino acid residues compared with the corre-sponding b chain, including g80 and g87 (Ada-chi and Asakura 1979; Nagel et al. 1979; Adachiet al. 1996). Hb D-Ibadan (b87 [F3] Thr . Lys),which introduces a lysine residue at the b87 po-sition, is presumed to have decreased interactionwith the mutant Val residue at HbSb6 (Watson-Williams et al. 1965; Nagel et al. 1979). Thus, HbD-Ibadan inhibits HbS polymerization. In con-trast, Hb Quebec-Chori (b87 [F3] Thr . Ile)was identified in a compound heterozygous pa-tient with mild to moderately severe sickle cellanemia (Witkowska et al. 1991). The introduc-tion of an isoleucine at b87 causes Hb Quebec-Chori to promote deoxygenated HbS polymer-ization.

Hb Bristol-Alesha (b67 [E11] Val . Met)(Molchanova et al. 1993; Rees et al. 1996) andHb Toms River (g67 [E11] Val . Met) (Crow-ley et al. 2011), which contain analogous aminoacid substitutions inb andg chains, respectively,represent interesting globin variants with mul-tiple biochemical defects. Hb Bristol-Alesha wasinitially observed in patients with moderatelysevere hemolytic anemia. Studies of the mutantprotein in patient erythrocytes revealed a b67Val . Asp substitution, predicted to renderthe protein unstable by introducing a highlycharged polar residue into the hydrophobic

heme pocket. However, subsequent DNA anal-ysis of the same patient identified a Val . Metcodon substitution (Rees et al. 1996). The inves-tigators concluded that the mutant Met residuewas converted to Asp posttranslationally, prob-ably through oxidative mechanisms. More re-cently, the analogous variant was identified infetal (g) globin (Hb Toms River) (Crowleyet al. 2011). The affected patient was a newbornpresenting with both cyanosis and anemia. DNAtesting revealed the codon change (Val . Met atE11). Mass spectrometry of patient hemolysateindicated a mixture of variantg globins contain-ing either Met or Asp at position E11. Althoughno structural studies have been performed withHb chains carrying Met or Asp E11, structureswith polar (Thr) or large aromatic (Trp) substi-tutions are available. These indicate that a rangeof amino acids can be accommodated withoutgross changes in the heme pocket structure (Fig.5B). Instead, altered steric and electrostatic in-teractions with the distal His and diatomic li-gands entering the heme pocket are likely to befunctionally significant. Biochemical studies in-dicated that the Hb Toms River Met substitutionproduced a stable, low O2 affinity variant g glo-bin, causing cyanosis. Its gradual posttransla-tional conversion to Asp destabilized the mole-cule, causing hemolytic anemia. This providesan example of how posttranslational modifica-tions of variant globins can modify phenotypes.The reason that Hb Bristol-Alesha causes pre-dominantly hemolytic anemia whereas HbToms River causes mainly cyanosis probably re-flects different rates of Met conversion to Asp inthe variant globin chains.

CONCLUDING REMARKS

More than 1000 Hb variants are known to exist(Globin Gene Server; Hardison et al. 2002; Giar-dine et al. 2011). These are mainly missensemutations that destabilize Hb, alter Hb-O2 af-finity, or most commonly, alter Hb functionminimally. Moreover, variants that do alter Hbbiochemistry are rarely life threatening or healthcompromising. Nonetheless, studies of thesevariants have been of great benefit to scienceand medicine for two main reasons. First,

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identification of Hb gene mutations as a causefor cyanosis, erythrocytosis, or mild hemolysisin otherwise healthy patients provides reassur-ance and minimizes additional diagnostic pro-cedures, sparing expense and risk. Second, ef-forts to understand how Hb variants producetheir structural, biochemical, and clinical effectshas generated important insights into red bloodcell function and also created general paradigmsfor the study of protein biology. Despite hun-dreds of studies over more than 50 years, new Hbvariants continue to emerge, yielding new in-sights into this important molecule.

ACKNOWLEDGMENTS

We thank Drs. Franklin Bunn, Kazuhiko Adachi,and John Olson for comments on the manu-script. Hemoglobin research in Dr. Weiss’s lab-oratory is funded through National Institutes ofHealth (NIH) grants DK061692, HL087427,and P30DK090969. The authors declare nocompeting financial interests.

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C.S. Thom et al.


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February 6, 20132013; doi: 10.1101/cshperspect.a011858 originally published onlineCold Spring Harb Perspect Med

Christopher S. Thom, Claire F. Dickson, David A. Gell and Mitchell J. Weiss Hemoglobin Variants: Biochemical Properties and Clinical Correlates

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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Hemoglobin Variants: Biochemical Properties and Clinical Correlatesperspectivesinmedicine.cshlp.org/content/3/3/a011858.full.pdf · Hemoglobin Variants: Biochemical Properties and