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Hemoglobin Variants: Biochemical Properties and 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 19104 3Menzies 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 acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochem- ical properties with physiological effects ranging from insignificant to severe. Studies of these mutations 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 have established important paradigms for the disciplines of structural biology, genetics, biochem- istry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

lobin gene mutations affecting hemoglobin stitutions, antitermination mutations, and al- G(Hb), the major blood oxygen (O2) carrier, tered posttranslational processing (Table 1). are common, affecting an estimated 7% of the Naturally occurring Hb mutations cause a world’s population (Weatherall and Clegg 2001; range of biochemical abnormalities, some of Kohne 2011). These mutations are broadly sub- which produce clinically significant symptoms. www.perspectivesinmedicine.org divided into those that impair globin protein The most common and medically important Hb subunit production (thalassemias) and those variants include HbS (Cao and Kan 2012; Lettre that produce structurally abnormal globin pro- 2012; Schechter and Elion 2012; Serjeant and teins (Hb variants). The latter class is mainly Rodgers 2012; Williams and Weatherall 2012), composed of missense mutations that cause sin- HbC (Cao and Kan 2012; Lettre 2012; Schechter gle amino acid substitutions in the globin pro- and Elion 2012; Serjeant and Rodgers 2012; tein, resulting in an abnormal, or “variant” Hb Williams and Weatherall 2012), HbE (see the tetramer. Less commonly, Hb variants are asso- sections on Selected Variants that Illustrate Im- ciated with deletions, multiple amino acid sub- portant Aspects of Hemoglobin Biology and

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C.S. Thom et al.

Table 1. Hb variants that are discussed in this article Globin site Amino acid Molecular Other biochemical Name (fold) substitution mechanism Clinical phenotype and laboratory findings Unstable Mutants Brockton b138 (H16) Ala . Pro Altered secondary Hemolytic anemia, structure reticulocytosis Philly b35 (C1) Tyr . Phe Altered a1b1 Hemolytic anemia, Decreased cooperativity, interface reticulocytosis increased oxygen affinity Peterborough b111 (G13) Val . Phe Altered a1b1 Hemolytic anemia, Decreased oxygen interface reticulocytosis affinity Stanmore b111 (G13) Val . Ala Altered a1b1 Hemolytic anemia Decreased oxygen interface affinity J-Guantanamo b128 (H6) Ala . Asp Altered a1b1 Hemolytic anemia Target cells interface Khartoum b124 (H2) Pro . Arg Altered a1b1 Normal interface Prato a1ora231 Arg . Ser Altered a1b1 Anisocytosis, Mildly unstable in (B12) interface hypochromia isopropanol Lombard a2 103 His . Tyr Altered a1b1 Anemia (G10) interface Contaldo a1ora2 His . Arg Altered a1b1 Hemolytic anemia 103 interface (G10) Foggia a2 117 Phe . Ser Altered a1b1 Microcytosis Rapidly degraded a (GH5) interface chains Groene Hart a1 119 Pro . Ser Altered a1b1 Hemolytic anemia, (H2) interface, microcytosis disrupted AHSP binding Turriff a1ora299 Lys . Glu Altered a1b1 Normal Comigrates with HbA1C, (G6) interface, rapidly degraded a disrupted AHSP chains binding Beziers a1 99 (G6) Lys . Asn Altered a1b1 Normal Comigrates with HbA1C interface, disrupted AHSP

www.perspectivesinmedicine.org binding Hirosaki a243 Phe . Leu Altered heme Heinz body hemolytic Hyperunstable (CE1) pocket anemia Terre Haute b106 (G8) Leu . Arg Altered heme Heinz body hemolytic Hyperunstable pocket anemia, dominant inclusion body thalassemia

High Affinity Variants Kempsey b99 (G1) Asp . Asn Unstable T state Erythrocytosis Decreased cooperativity Hiroshima b146 His . Asp Mutated Bohr Erythrocytosis Decreased cooperativity, (HC3) proton donor decreased Bohr effect York b146 His . Pro Mutated Bohr Erythrocytosis Decreased cooperativity, (HC3) proton donor decreased Bohr effect Cowtown b146 His . Leu Mutated Bohr Erythrocytosis Decreased Bohr effect (HC3) proton donor Continued

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Hemoglobin Variants

Table 1. Continued Globin site Amino acid Molecular Other biochemical Name (fold) substitution mechanism Clinical phenotype and laboratory findings Rahere b82 (EF6) Lys . Thr Altered 2,3DPG Erythrocytosis binding site Providence b82 (EF6) Lys . Asn Altered 2,3DPG Erythrocytosis Low oxygen affinity binding site Helsinki b82 (EF6) Lys . Met Altered 2,3DPG Erythrocytosis Decreased Bohr effect binding site

Low Affinity Variants Kansas b102 (G4) Asn . Thr Unstable R state Cyanosis Decreased cooperativity Beth Israel b102 (G4) Asn . Ser Unstable R state Cyanosis Decreased cooperativity St. Mande´ b102 (G4) Asn . Tyr Unstable R state Cyanosis

Methemoglobin Variants M-Iwate a1ora2 His . Tyr Oxidized heme Pseudocyanosis Abnormal visible (F8) (Methemoglobinemia) spectrum M-Saskatoon b63 (E7) His . Tyr Oxidized heme Pseudocyanosis Abnormal visible (Methemoglobinemia) spectrum

Globin Chain Elongation Variants Constant a2 142 Stop . Antitermination Microcytosis Decreased mRNA Spring (HC3) Gln mutant stability Cranston b145 þCT Frameshift, Hemolytic anemia Increased oxygen (HC3) elongated affinity, decreased globin cooperativity

Variants with Multiple Effects HbE b26 (B8) Glu . Lys Unstable, reduced Microcytosis synthesis Bruxelles b41 (C7) or Phe . 0 Altered heme Hemolytic anemia, Heinz bodies, decreased b42 pocket cyanosis, cooperativity (CD1) splenomegaly, reticulocytosis www.perspectivesinmedicine.org Warsaw b42 (CD1) Phe . Val Altered heme Hemolytic anemia, Heinz bodies, pocket cyanosis decreased cooperativity Hammersmith b42 (CD1) Phe . Ser Altered heme Hemolytic anemia, Heinz bodies, pocket cyanosis decreased cooperativity Buccuresti- b42 (CD1) Phe . Leu Altered heme Hemolytic anemia, Heinz bodies, Louisville pocket cyanosis decreased cooperativity Zurich b63 (E7) His . Arg Altered heme Normal, but Decreased cooperativity, pocket hypersensitive to increased oxygen oxidative stress affinity, increased CO affinity Jamaica Plain b6 (A3) Glu . Val Altered secondary Hemolytic anemia, Unstable, Heinz bodies, and b68 and Leu structure cyanosis, sickle cell phenotype, (E12) . Phe splenomegaly, splenic decreased oxygen sequestration affinity Continued

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Table 1. Continued Globin site Amino acid Molecular Other biochemical Name (fold) substitution mechanism Clinical phenotype and laboratory findings Quebec-Chori b87 (F3) Thr . Ile Altered interaction Normal Promotes HbS with HbS polymerization polymer D-Ibadan b87 (F3) Thr . Lys Altered interaction Normal Inhibits HbS with HbS polymerization polymer Bristol-Alesha b67 (E11) Val . Met Altered heme Hemolytic anemia, Heinz bodies, pocket reticulocytosis decreased cooperativity, decreased Bohr effect, decreased oxygen affinity Toms River g67 (E11) Val . Met Altered heme Anemia, cyanosis Unstable, low oxygen pocket affinity 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).

Variantsthat Affect Multiple Hemoglobin Func- sive reviews of this topic, see Nathan and Oski’s tions; see also Musallam et al. 2012), and some Hematology of Infancy and Childhood (Nathan thalassemias (e.g., “thalassemic hemoglobinop- et al. 2009); Hemoglobin: Molecular, Genetic, athies”), all of which are under positive genetic and Clinical Effects (Bunn and Forget 1986); selection because they confer survival advan- Disorders of Hemoglobin: Genetics, Pathophysiol- tages in areas where malaria is endemic (Weath- ogy, and Clinical Management (Steinberg et al. erall and Clegg 2001). In addition to these prev- 2001); and the Globin Gene database (Hardison alent mutant proteins, there are also .1000 et al. 2002; Giardine et al. 2011). other known naturally occurring Hb variants, which are rare individually but common collec- tively. Most Hb mutants are cataloged on the BASIC PRINCIPLES Globin Gene database (HbVar, http://globin. Hemoglobin Synthesis, Structure, and bx.psu.edu; Hardison et al. 2002; Giardine et

www.perspectivesinmedicine.org Function al. 2011). By convention, these variants are named after the geographic origin of the affect- Hemoglobin is a heterotetramer composed of ed individual. Although many Hb variants are a-like and b-like globin subunits, each bound clinically silent, some produce clinical manifes- to a heme prosthetic group. The major functions tations of varying severity. Analyses of these of Hb are to transport oxygen (O2) from the variants, which can be considered to be “exper- lungs to peripheral tissues and carbon dioxide iments of nature” (Garrod 1928), have generated (CO2) from the tissues to the lungs. The kinetics valuable insights into structure–function rela- of Hb-O2 binding and release are fine-tuned for tionships within the Hb molecule, with interest- this purpose and adaptable according to devel- ing and important clinical consequences. opmental ontogeny and metabolic perturba- The goal of this work is to provide a succinct tions. Moreover, the Hb molecule must limit po- conceptual framework for understanding the tential problems caused by its associated iron biology and clinical implications of Hb vari- and free O2, reactivemolecules capable of inflict- ants. We explore major concepts of Hb biology ing damage through the production of reactive followed by a discussion of selected Hb variants oxygen species. Efforts to understand how Hb that reinforce basic principles. For more exten- structure imparts these critical functions have

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Hemoglobin Variants

been ongoing for more than 50 years. Hemoglo- globin (metHb) to its reduced form. Not sur- bin was one of the first proteins to be sequenced prisingly, globin mutations that alter amino (Konigsberg et al. 1961; Schroeder et al. 1961; acids within the ligand pocket frequently pro- Watson and Kendrew 1961) and the globin genes duce strong functional effects, including desta- were among the earliest to be cloned (Rabbitts bilization, altered affinity for O2, and increased 1976). In the late 1950s, Perutz and colleagues rates of metHb formation and heme loss (see determined the three-dimensional structure of the section on Selected Variants that Illustrate Hb through X-ray crystallography (Perutz 1960; Important Aspects of Hemoglobin Biology). Perutz et al. 1960). More recent studies refined Variants promoting autoxidation are termed this structure to high resolution (Paoli et al. “M-Hbs” (see the sections on Selected Variants 1996; Parket al. 2006). In addition, O2 and other that Illustrate Important Aspects of Hemoglo- ligand-binding properties have been measured bin Biology and Methemoglobin (“M-Type”) in detail for native Hb and many naturallyoccur- Variants). Because Hb gains its distinctive color ring mutants. All of this work provides a sub- from the heme group, alterations that affect stantial framework for defining the O2 delivery the environment of the heme iron, including properties of Hb, as well as the molecular conse- changes in the surrounding amino acids, dif- quences of variant mutations (see, for example, ferent gas ligands, or redox state, produce char- Lehmann 1957; Konigsberg and Lehmann 1965; acteristic changes in visible light absorption. Shimizu et al. 1965; Perutz and Lehmann 1968; These color changes are used clinically to assess Perutz 1970; Bunn and Forget 1986). Hb-O2 saturation, metHb formation and the Globin polypeptides are synthesized from effects of Hb variants. separate a-like and b-like globin gene clusters Within the Hb tetramer, each globin subunit located on human chromosomes 16 and 11, binds the unlike chain through two distinct in- respectively. Nascent globin chains rapidly in- terfaces, termed a1b1 and a1b2 (Fig. 1D). In- corporate heme, which stabilizes their native dividual globin subunits form dimers through folding into Hb subunits composed of seven the extremely high affinity a1b1 interaction. or eight a helices named A–H, which fold to- Globin chain monomers are relatively unstable gether into a globular structure (Fig. 1A). Hb compared with dimers, with a tendency to form subunits bind O2 and other ligands via the heme intracellular precipitates that damage erythro- iron buried within an evolutionarily conserved cytes, causing hemolytic anemia. Thus, muta- hydrophobic pocket that faces the outside of tions that impair the a1b1 interaction can cause HbA tetramers (Fig. 1B). Heme iron is axially erythrotoxicity by favoring the accumulation coordinated to globin proteins by an invariant of monomeric subunits (Fig. 2C). The a1b2 in- www.perspectivesinmedicine.org histidine residue in helix F8, termed the “prox- teraction, which is lower affinity, mediates imal” histidine (Fig. 1C). The opposite axial tetramerization. Oxygen binding destabilizes position binds O2, which is stabilized by inter- the a1b2 interaction, resulting in a transition action with the conserved “distal” histidine in of the quaternary structure from the “T” (tense, helix E7 (Fig. 1C). Multiple additional amino low affinity, deoxygenated) to “R” (relaxed, high acids within the globin proteins stabilize heme affinity, oxygenated) state, which facilitates O2 binding through noncovalent interactions (Fig. binding to additional subunits (Fig. 2A). This 2þ 1C). Iron must be in its reduced (ferrous, Fe ) process causes cooperative O2 binding, which state for Hb to bind O2. Oxidized or “met” Hb is illustrated by the characteristic sigmoidal 3þ (ferric, Fe ) cannot bind O2 and is relatively shape of the Hb-O2 equilibrium curve (Fig. unstable, tending to lose hemin and denature. 2B). Cooperativity allows maximal O2 release Thus, red blood cells have evolved elaborate over relatively small drops in O2 tension. Muta- mechanisms to maintain Hb in its reduced state tions within the a1b2 interaction region can al- (Bunn and Forget 1986; Ganz and Nemeth ter the functional properties of Hb, mainly by 2012; Schechter 2012). For example, the methe- perturbing O2-binding characteristics (Fig. 2C, moglobin reductase system converts methemo- cyan spheres; Table 1).

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A D(β) B α1 α2

A B C Heme G E

β β F 1 2 H

His E7 His E7 C Phe CD1 Phe CD1

Val E11 Val E11

Heme Heme

His F8 His F8

β D α β α 2 2

α1β2 β interface

β1 α1

α1β1 α interface

Figure 1. The structure of Hb. (A) The a (pink) and b (red) Hb subunits have conserved a-helical folds. Helices

www.perspectivesinmedicine.org are labeled A–H from the amino terminus. The a subunit lacks helix D. (B) The high O2 affinity R state quaternary structure of Hb with O2 (red spheres) bound at all four heme sites (protoporphyrin-IX as yellow sticks, with central iron atom as orange sphere). (C) Stereo (wall-eye) diagram of the heme pocket of b showing the proximal (F8) and distal (E7) histidines and selected residues in the distal heme pocket that influence ligand binding and autoxidation. (D) Hb tetramer is assembled from two identical ab dimers (shown in red and gray for 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).

Cooperativity represents a general phenom- Bohr effect (Perutz et al. 1984). In peripheral enon termed allosteric regulation, in which ef- tissues, abundant CO2 is taken up by red blood fector molecules control the properties of en- cells and metabolized by carbonic anhydrase to zymes or other proteins by binding to regions carbonic acid, resulting in the production of Hþ that are distinct from the active sites. Several al- and consequent O2 release. In contrast, relatively losteric regulators, in addition to O2, influence low CO2 and high pH in the lung favors O2 bind- þ the properties of Hb. For example, H binds Hb ing to Hb. Additionally, CO2 forms carbamino to promote O2 release in a process termed the adducts with the amino termini of both a and b

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High O2 affinity

dacdOln ril.Ct hsatceas article this Cite Article. Online Advanced 100 A B Hb variants 90 pH α (oxy) 2,3DPG α (deoxy) 2 80 2 β (deoxy) β Temp 2 2(oxy) 70 60 Low O2 affinity Hb variants α β pH 1 2 50 2,3DPG interface 40 Temp Saturation (%) Saturation 2

O 30 20 β α β 1 1 1 α1β1 10 0 interface 0 10 20 30 40 50 60 70 80 90 100

pO2(mm Hg)

C D onOctober4,2021-PublishedbyColdSpringHarborLaboratoryPress odSrn abPrpc Med Perspect Harb Spring Cold K11E (Turriff) K11N (Beziers) H146D (Hiroshima) H146P (York) H103Y (Lombard) H103Y (Lombard) AHSP H146L (Cowtown) H103R (Contaldo) H103R (Contaldo) 2,3DPG D99N (Kempsey) A128D (J-Guantanamo) R31S (Prato) K82M (Helsinki) K82N (Providence) R31S (Prato) α K82T (Rahere) β α o:10.1101 doi: N102T (Kansas) 1 1 N102S (Beth Israel) N102Y (St. Mandé) P124R (Khartoum) Y35F (Philly) F117S (Foggia)

P119S (Groene Hart) F117S (Foggia) / cshperspect.a011858 V111F (Peterborough) P119S (Groene Hart)

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 from Variants Hemoglobin the deoxy (orange) to the oxy (red) states. (B) The sigmoidal shape of the Hb-O2 saturation curve shows allosteric regulation by changes in pH, temperature, and 2,3DPG. These regulators, as well as Hb variants, influence the shape of the curve. High oxygen affinity variants, high pH, low 2,3DPG, or low temperature induce a “left shift” in the saturation curve (red line). Conversely, low oxygen affinity variants, low pH, high 2,3DPG, or high temperature induce a “right shift” (blue line). (C) Hb sequence variants at the allosteric a1b2 interface (cyan spheres) show an impaired response to O2 binding. Some sequence variants disrupt binding to other allosteric regulators, e.g., substitutions at bK82 (green) disrupt interactions with 2,3DPG that normally stabilize the low O2 affinity Tstate. Mutations that disrupt a1b1 (and a2b2) dimerization (blue spheres) increase the concentration of free monomers, 7 which are unstable. (D) Some a mutations that disrupt binding to b may also disturb binding to the chaperone, AHSP.Other a variants, such as Turriff and Beziers (pink sphere) may inhibit only AHSP binding. Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press

C.S. Thom et al.

globins in vivo. These interactions produce mi- also cause blue-tinged skin, whereas the blood nor effects on whole blood O2 affinity compared itself appears brown. Studies of one family with to the impact of CO2 on pH (Bunn and Forget congenital cyanosis led Horlein and Weber 1986). The compound 2,3-diphosphoglycerate to describe the first known hemoglobinopathy, (2,3DPG), formed as a by-product of glycolysis caused by the variant Hb-M Saskatoon (Horlein and present at relatively high concentrations in and Weber 1948). The M-Hb variant Iwate, red blood cells, is another important allosteric whichcauses“blackblood”or“hereditarynigre- regulator of Hb. 2,3DPG binds and stabilizes mia,”was first described more than 200 years ago T state Hb to facilitate O2 release. Hemoglobin in Japan (Shibata et al. 1960). bindingsitesforHþ and2,3DPGhavebeeniden- Itisimportant tonote thatmanyHb variants tified (Perutz et al. 1969, 1984; Perutz 1970; Ar- affecting skin color are not clinically damag- none 1972). Together, these interactions allow ingbeyondtheircosmeticeffects.However,these Hb to sense metabolic activity and modulate conditions can mimic more life-threatening O2 binding accordingly. This environmental problems such as cardiopulmonary and myelo- sensory function can be altered by mutations proliferative disorders, which must be excluded. that affect the affinity of Hb for its allosteric reg- Thus,patientswithcyanoticorpolycythemicHb ulators (Fig. 2C, green sphere; Table 1). variants may mistakenly undergo unnecessary and potentially dangerous medical procedures. Historical examples include cyanotic patients Identification of Hb Variants and Their undergoing surgery or catheterization for pre- Clinical Implications sumed heart defects and polycythemic patients Many Hb variants are readily ascertained receiving radioactive 32P for presumed polycy- through physical examination and/or routine themia vera (Steinberg et al. 2001; Nathan et al. laboratory testing, which explains why so many 2009). As stated, “the primary reason for estab- have been discovered. Uncommonly, some vari- lishing the diagnosis [of M Hbs] is to prevent ants are identified through evaluation of ill pa- iatrogenic misadventures that might arise under tients with severe anemia orclinically significant the mistaken impression that the patient has a cyanosis. Many amino acid substitutions alter cardiac or pulmonary disorder” (Bunn and For- surface charge and are thus detected by electro- get 1986). Most Hb variants with altered O2 af- phoresis or chromatography, techniques which finity are relatively simple to diagnose through are routinely performed on neonates in North history, physical examination, and laboratory America and Europe. Interestingly, afew Hb var- testing (Wajcmanet al. 2001; Wajcmanand Mo- iants migrate similarly to HbA1c, a glycosylated radkhani 2011). www.perspectivesinmedicine.org form of Hb that reflects long-term control of blood glucose levels in diabetic patients (Table Globin Gene Synthesis Is Developmentally 1) (reviewed in Little and Roberts 2009). In this Regulated way, specific Hb amino acid substitutions can artificially elevate HbA1c measurement and in- Developmental regulation of the a-like and b- terfere with diabetic management. Other Hb like globin gene families is of great medical variants are clinically benign but produce obvi- significance (Sankaran and Orkin 2012). Hemo- ous changes in skin color. For example, muta- globin F (a2g2) is the most highly expressed tions that increase Hb-O2 affinity typically stim- form during late fetal gestation. After birth, ex- ulate erythropoietic drive by inhibiting O2 tissue pression gradually switches to HbA (a2b2)over delivery, causing erythrocytosis associated with several months. Thus, symptomatic mutations a ruddy complexion (Nathan et al. 2009). Mu- affecting a or g globins are present prenatally or tations that reduce O2 affinity produce a bluish at birth, whereas the manifestations of b-glo- hue to the skin (cyanosis) caused by abnormal- bin mutations are typically delayed until a few ly high levels of deoxyHb. Mutations that favor months after birth. Interestingly, g-globin gene oxidation of Hb iron to the met form (M-Hbs), mutations that are apparent at birth, most

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Hemoglobin Variants

typically reflected by cyanosis or hemolytic ane- testing more typically uses isoelectric focus- mia (e.g., Hb-F TomsRiver, sections on Selected ing and HPLC, which are more sensitive. Variants that Illustrate Important Aspects of b. Hb-O binding curve. This test, performed Hemoglobin Biology and Variants that Affect 2 on whole red blood cells or hemolysate, in- Multiple Hemoglobin Functions, and Table 1), dicates the percent (%) oxygenated Hb at a fade over a few weeks as Hb production switches given O2 partial pressure (Fig. 2B). Hemo- from F (a2g2)toA(a2b2). globin variants with an abnormally high O2 affinity (see sections on Selected Variants Laboratory Testing for Hb Variants that Illustrate Important Aspects of Hemo- globin Biology and High Oxygen Affinity In many countries, routine testing of all new- Variants) will become saturated at lower borns is performed to identify common hemo- O pressures producing a “left-shifted” O globinopathies such as some thalassemias and 2 2 equilibrium curve, whereas mutations that HbS. Isoelectric focusing or high-performance reduce O affinity (see sections on Selected liquid chromatography (HPLC), the most com- 2 Variants that Illustrate Important Aspects monly used tests, identify most structurally ab- of Hemoglobin Biology and Low Oxygen normal Hbs (Wajcman et al. 2001). In this way, Affinity Variants) will cause the opposite many benign Hb variants are discovered inci- “right shift.” Determination of Hb-O af- dentally. Clinical indications for laboratory test- 2 finity responses to allosteric regulators, par- ing to investigate potential Hb variants are listed ticularly 2,3DPG or Hþ (pH changes), in Table 2. can provide insight into the structural caus- Specific laboratory tests to investigate Hb es of the observed phenotypes. Unfortu- variants include: nately, few clinical laboratories currentlyof- a. Physical methods of Hb separation. These fer this assay. include electrophoretic and chromato- c. Visible wavelength spectroscopy. Hemoglo- graphic techniques that examine the physi- bin variants with amino acid substitutions cal properties of a1b1 dimers or individual in the heme pocket affect visible light ab- globin subunits. Specific standards, such sorbance. For example, M-type Hbs show as HbA, HbS, HbC, HbF, and HbA ,are 2 characteristic spectra that can distinguish typically examined as controls. Hb variants them from methemoglobinemia caused by may show altered migration in these assays. an enzyme deficiency in the metHb reduc- Historically, cellulose acetate and citrate tase system (Bunn and Forget 1986; Dailey

www.perspectivesinmedicine.org agar electrophoresis were most commonly and Meissner 2012; Ganz and Nemeth used to detect variant Hbs. Current clinical 2012; Schechter 2012). Pulse oximetry is a noninvasive spectrophotometric test that Table 2. Clinical indications for laboratory testing to measures absorbance ratios at specific diagnose Hb variants wavelengths for oxygenated (660 nm) and Indications for hemoglobin testing deoxygenated (940 nm) blood (Tremper Routine newborn testing for common and Barker 1989). This can produce confus- hemoglobinopathies (i.e., HbS, HbC, ing and sometimes misleading results in pa- thalassemias) tients with variant Hbs that show unique Cyanosis with adequate arterial oxygenation and no light absorbance properties (Verhovsek et apparent cardiopulmonary disease al. 2010). In these cases, analysis of arteri- Erythrocytosis with normal or elevated al blood O2 concentration may be required erythropoietin levels to rule out hypoxia. Analyzing these variant Unexplained hemolytic anemia Hbs using light absorbance throughout the Unexplained thalassemia phenotype full visible wavelength spectrum can pro- Family history consistent with an Hb variant vide useful diagnostic information.

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C.S. Thom et al.

d. Hemoglobin stability testing. Typically, Hb that destabilize Hbs: amino acid substitutions stability is impaired in variants that are withinthehemepocket,disruption ofsecondary associated with hemolytic anemia. Hemo- structure, substitution in the hydrophobic inte- globin stability tests measure the propensity rior of the subunit, amino acid deletions, and for Hb to denature on exposure to various elongation of the subunit (Bunn and Forget stresses including heat (Carrell and Kay 1986). 1972), isopropanol (Bender et al. 1981), More than 75% of Hb is a helical (Perutz mechanical agitation (Asakura et al. 1975), et al. 1960; Park et al. 2006). This structure is and zinc acetate (Roth et al. 1976). The particularly susceptible to disruptions by pro- Heinz body test uses supravital stains, line substitutions (Levitt 1981). For example, such as methylene blue or crystal violet, in Hb Brockton (b138 [H16] Ala . Pro) the to detect aggregated globins within ery- substituted proline disrupts intermolecular hy- throcytes (Eisinger et al. 1985). drogen bonding between b138Ala and b134Val in helix H (Russu and Ho 1986; Moo-Penn et al. e. Specialized testing. Mass spectrometry anal- 1988). This produces an unstable variant with a ysis of patient hemolysate and DNA se- propensity to precipitate and aggregate, there- quencing of globin genes are specialized by damaging erythrocytes and predisposing to confirmatory tests to identify amino acid hemolysis. Hb Brockton does not show altered and nucleotide alterations associated with O binding affinity or electrophoretic mobility suspected Hb variants (Wajcman et al. 2 shifts. This variant was identified by HPLC anal- 2001; Wajcman and Moradkhani 2011). ysis of patient globin chains and its altered X-ray DNA sequencing may readily elucidate the crystallography diffraction pattern shows local presence of an Hb variant. However, addi- disruption of helix H (Moo-Penn et al. 1988). tional biochemical and structural studies, Mutations at the a1b1 interface can cause including those discussed in this section, hemolytic anemia by inhibiting heterodimer are required to determine how the variant formation, favoring the accumulation of free affects Hb function. Crystallographic anal- globin subunits, which themselves are unstable, ysis is the highest resolution approach to particularly a chains (Fig. 2C, blue spheres). Ex- determine the effects of globin amino acid amples include Hb Philly (b35 [C1] Tyr . Phe) substitutions on molecular structure. Crys- (Rieder et al. 1969), Hb Peterborough (b111 tallography has been used historically as a [G13] Val . Phe) (King et al. 1972), Hb Stan- research tool to assess the effects of some more (b111 [G13] Val . Ala) (Como et al. interesting Hb variants (see, for example, 1991), and Hb J-Guantanamo (b128 [H6] Ala

www.perspectivesinmedicine.org Pulsinelli et al. 1973; Perutz et al. 1984). . Asp) (Martı´nez et al. 1977). Hb Khartoum (b124 [H2] Pro . Arg) contains a substitution SELECTED VARIANTS THAT ILLUSTRATE at the a1b1 interface that is mildly destabilizing IMPORTANT ASPECTS OF HEMOGLOBIN in vitro, but does not cause clinical symptoms BIOLOGY (Clegg et al. 1969; Argos et al. 1979). Interestingly, some a-globin (HBA) gene Unstable Variants mutations affecting the a1b1 interface may Unstable variants frequently cause congenital also destabilize free a chains by inhibiting their Heinz body hemolytic anemia detected by labo- binding to a-hemoglobin stabilizing protein ratory screening and clinical symptoms (see sec- (AHSP), an erythroid molecular chaperone tions on Basic Principles and Laboratory Testing that facilitates Hb assembly (Fig. 2D, blue for Hb Variants and Table 2). Mutations that spheres) (reviewed in Weiss et al. 2005; Favero alter any step in globin processing, including and Costa 2011). These a-globin variants in- subunit folding, heme interaction, dimeriza- clude Hbs Prato (a1ora2 31 [B12] Arg . tion, or tetramerization, can destabilize Hb. Ser) (Marinucci et al. 1979), Lombard (a2 103 Bunn and Forget note five general mechanisms [G10] His . Tyr) (Hoyer et al. 2002), Contaldo

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Hemoglobin Variants

(a1ora2103[G10] His . Arg) (Sciarrattaetal. as Heinz bodies. Missense mutations also cause 1984), Foggia (a2 117 [GH5] Phe . Ser) (La- hyperunstable Hb variants. Hb Hirosaki (a243 cerraetal. 2008),Groene Hart (a1119[H2] Pro [CE1] Phe . Leu) was discovered in a family . Ser) (Harteveld et al. 2002; Vasseur-Godbil- with hemolytic anemia (Ohba et al. 1975; Tana- lon et al. 2006; Giordano et al. 2007; Vasseuret al. ka et al. 2005). After several tests failed to iden- 2009), and others (Wajcmanet al. 2008; Yu et al. tify a soluble variant Hb within erythrocytes, 2009). Naturally occurring a-globin variants DNA sequencing was used to characterize the with amino acid substitutions at position 99 in- mutation. Hb Terre Haute (b106 [G8] Leu . cludingHb Turriff(a1ora299[G6]Lys . Glu) Arg) is another hyperunstable variant associated (Langdown et al. 1992) and Hb Beziers (a199 with a severe Heinz body hemolytic anemia and [G6] Lys . Asn) (Lacan et al. 2004) bind b glo- globin chain imbalance (Coleman et al. 1991). bin normally but show impaired interaction In initial studies of patient erythroid cells, per- with AHSP and may be mildly destabilizing formed in 1979, abnormal Hb tetramers were (Fig. 2D, violet spheres) (see also Yu et al. 2009; not detected and peptide mapping of radiola- Khandros et al. 2012; Mollan et al. 2012). beled nascent globins identified a b112 (G14) Antitermination mutations can also destabilize Cys . Arg substitution, originally termed Hb a globin in part by impairing its binding to Indianapolis (Adams et al. 1979). However, the AHSP (Turbpaiboon et al. 2006). b112 Cys . Arg mutation was subsequently Hyperunstable Hb variants precipitate identified in unrelated individuals with much shortly after synthesis and are not incorporated less severe disease. In 1991, reevaluation of the into Hb tetramers. These ephemeral proteins original pedigree by DNA analysis discovered a are difficult to isolate. In this case, electropho- b106 Leu . Arg mutation, which was renamed resis may be falsely negative due to the rapid Hb Terre Haute (Coleman et al. 1991). Most turnover of these variants, making DNA se- likely, incomplete tryptic cleavage of the abnor- quencing a critical diagnostic test. Affected pa- mal b-globin peptide in the earlier studies led to tients show a dominantly inherited “inclusion misidentification of the causative mutation. body thalassemia” resulting from both the pre- This work reflectsthe interesting historical point cipitated variant globin and consequent chain that many Hb variants were identified through imbalance with accumulation of the unaffected laborious and technically challenging protein globin, which is unstable in its free form (Sta- studies performed some time ago, before DNA matoyannopoulos et al. 1974). Patient erythro- sequence analysis of patient globin genes was cytes typically display abnormal morphology feasible. Reevaluation of these mutations with microcytosis, hypochromia, moderate to through genetic testing has yielded some inter- www.perspectivesinmedicine.org severe anisopoikilocytosis, basophilic stippling, esting surprises (see also the discussion on Hb and inclusions that may be become particularly Bristol-Alesha and the section on Variants that prominent following splenectomy (Steinberg Affect Multiple Hemoglobin Functions). et al. 2001). Weatherall, Thein, and colleagues characterized several hyperunstable mutations High Oxygen Affinity Variants in exon 3 of the b-globin gene (Thein et al. 1990). All of the mutations were frameshifts Hemoglobin variants with increased O2 affinity or nonsense codons that produced relatively cause erythrocytosis by stimulating erythro- long (.120 amino acid) proteins with car- poietic drive (see Hebbel et al. 1978 for a de- boxy-terminal truncations. The investigators scription of the associated physiology). This proposed that truncated globins causing dom- commonly results from amino acid substitu- inantly inherited thalassemia are long enough tions that stabilize the R (high O2 affinity) state to bind heme posttranslationally, which ren- relative to the T (low O2 affinity) state and/or dered them relatively resistant to proteolytic inhibit responses to environmental allosteric degradation, allowing for subsequent precip- regulators that stimulate O2 release, including itation of heme-containing aggregates detected Hþ (Bohr effect) or 2,3DPG (see the sections on

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Basic Principles and Hemoglobin Synthesis, within these regions cause high O2 affinity vari- Structure, and Function). Because T to R state ants. In addition, b146 His at the carboxyl ter- transitions are mediated largely through a1b2 minus contributes significantly to the Bohr ef- interactions, high affinity variants frequently re- fect by forming a salt bridge with b94 Asp sult from substitutions that alter this interface (Perutz et al. 1984). This interaction is disrupted (Fig. 2C, cyan spheres). For example, the amino in several high O2 affinity variants with b146 acid replacementin Hb Kempsey(b99 [G1] Asp substitutions: Hb Hiroshima (b146 [HC3] His . Asn) perturbs a1b2 interactions by prevent- . Asp) (Hamilton et al. 1969; Perutz et al. 1971; ing the formation of a hydrogen bond between Imaietal.1972;Olsonetal.1972),HbYork(b146 b99 Asp and a42 Tyr, which normally stabilizes [HC3] His . Pro) (Bare et al. 1976), and Hb the deoxygenated low O2 affinity Tstate (Fig. 3) Cowtown (b146 [HC3] His . Leu) (Fig. 2C) (Reed et al. 1968; Lindstrom et al. 1973; Bunn (Schneider et al. 1979; Perutz et al. 1984). These et al. 1974). This structural change shifts quater- variants show reduced Bohr effect with im- naryequilibrium toward the oxygenated R form, paired release of O2 under acidic conditions. which impairs O2 release to peripheral tissues Several high affinity Hb variants are caused and thus increases erythropoietic drive. The car- by substitutions that inhibit interaction with boxyl termini of globin chains are also involv- 2,3DPG, which normally binds globin chains ed in a1b2 interactions that stabilize the low to stimulate O2 release (Fig. 2C). For example, O2 affinity T state and numerous substitutions Hb Rahere (b82 [EF6] Lys . Thr) replaces an

A α α 1 2

B β β 1 2 www.perspectivesinmedicine.org α α Y42 1 Y42 αD99N 1 W37 W37 (Kempsey) N97 N97 D94 D94 D99 D99 N102 N102 αN102T β (Kansas) β 2 2 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 of the figure show an overlay of the R-state (red/pink) and T-state (orange/light orange) quaternary structures of Hb. The allosteric a1b2 interface is boxed. (B) Detail of the a1b2 interface in the deoxy T state (b2 chain in orange, 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 residues affect only one state. Mutation of Asp99 to Asn (Hb Kempsey) compromises electrostatic interactions in the deoxygenated 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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Hemoglobin Variants

invariant lysine in the 2,3DPG binding site of inhibits the formation of a hydrogen bond b globin, thereby reducing the affinity for this with Asp94 that normally stabilizes the oxygen- allosteric regulator (Lorkin et al. 1975; Sugi- ated R structure. A similar mechanism causes hara et al. 1985). Consequently, Hb Rahere low O2 affinity in two other Hb variants through shows blunted O2 release in response to added different substitutions of the same amino acid 2,3DPG in vitro. In vivo, O2 release in peripheral residue (B102 [G4] Asn) in Hb Beth Israel (b102 tissues is inhibited, resulting in elevated blood [G4] Asn . Ser) (Nagel et al. 1976) and Hb Hb levels. Similarly, Hb Providence (b82 [EF6] St. Mande´ (b102 [G4] Asn . Tyr) (Arous et Lys . Asn) (Bonaventura et al. 1976; Moo- al. 1981; Poyart et al. 1990). Penn et al. 1976) and Hb Helsinki (b82 [EF6] Lys . Met) (Ikkala et al. 1976; Charache et al. Methemoglobin (“M-Type”) Variants 1977) are high O2 variants caused by different amino acid substitutions at the 2,3DPG binding Hemoglobin iron must be in its reduced (Fe2þ, site on b82. ferrous) state to bind O2. Moreover, oxidized (Fe3þ, ferric, met) Hb is intrinsically unstable with a tendency to release heme. Hemoglobin Low Oxygen Affinity Variants reduction is maintained through intrinsic fea- Low O2 affinity Hb variants typically present tures of the Hb protein and extrinsic antioxi- with cyanosis. In general, these variants are dant pathways within red blood cells (see the caused by globin amino acid substitutions that sections on Basic Principles and Hemoglobin tip the quaternary equilibrium of Hb tetramers Synthesis, Structure, and Function). Exposure from the high affinity oxygenated R state to the to oxidant drugs or toxins, genetic alterations low affinity deoxygenated T state; more or less in erythroid metHb reductase enzyme systems the opposite of what occurs for high O2 affinity (Ganz and Nemeth 2012; Schechter 2012), or variants (see the sections on Selected Variants globin chain variants can predispose to met- that Illustrate Important Aspects of Hemoglo- hemoglobinemia. These disorders present as bin Biologyand High Oxygen Affinity Variants). “pseudocyanosis,” (i.e., low Hb-O2 saturation), This does not inhibit Hb-O2 release in tis- despite adequate arterial oxygenation. Detailed sue capillaries, but rather, interferes with Hb- in vitro analyses of the red cell and isolated Hb O2 uptake if the P50 has increased to 50 mm samples can usually distinguish wild-type Hg. Paradoxically, low O2 affinity Hb variants metHb resulting from toxins or defective reduc- can be associated with mild anemia thought to tase systems and M-type Hb variants that are be caused by increased O2 tissue delivery with predisposed to spontaneous oxidation (Bunn www.perspectivesinmedicine.org reduced erythropoietic drive (Stamatoyanno- and Forget 1986; Steinberg et al. 2001; Nathan poulos et al. 1969). In addition, some low O2 et al. 2009). For example, various M-Hbs show affinity mutants are unstable and therefore as- characteristic visible absorbance spectra. sociated with not only cyanosis but also Heinz Globin variants associated with metHb for- body hemolytic anemia (see the sections on Se- mation are typically caused by amino acid sub- lected Variants that Illustrate Important Aspects stitutions within the heme pocket. For example, of Hemoglobin Biology and Unstable Variants). four different M-Hbs occur when tyrosine re- Several low affinity variants involve replace- places the a-orb-globin proximal or distal his- ments at the a1b2 interface, which plays an tidine residues that interact with heme (Fig. 1C) important role in Hb cooperativity. Hb Kansas (reviewed in Adachi et al. 2011). In Hb M-Iwate (b102 [G4] Asn . Thr) is awell-studied low O2 (a1ora2 87 [F8] His . Tyr), the proximal affinity variant (Fig. 3B) (Reissmann et al. 1961; histidine is replaced by tyrosine (Fig. 4A), which Bonaventura and Riggs 1968; Gibson et al. 1973; deprotonates and coordinates to the heme iron Riggs and Gibson 1973). Affected individuals (Fig. 4B) (Konigsberg and Lehmann 1965; Shi- are markedly cyanotic, although clinically well. mizu et al. 1965). Ferric heme, bound through Replacement of Asn102 at the a1b2 interface the native His F8, is readily reduced by metHb

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C.S. Thom et al.

reductase (Fig. 4C). Tyrosine (F8) coordination variant, the protonated form of mutant Tyr can stabilizes the oxidized ferric state and decreases bind ferric heme iron to generate a hexacoordi- reactivity with metHb reductases. This interac- nate structure that is relatively easily reduced by tion also distorts the position of heme and helix cellular metHb reductases (Fig. 4E). Conse- F within the altered a subunits. In native Hb, quently, patients with Hb M-Saskatoon have movement of the proximal His F8 and F helix lower levels of circulating oxidized Hb com- away from the heme group stabilizes the deox- pared with those with Hb M-Iwate. Compara- ygenated T state and reduces the O2 affinity of tive studies of these patients and their variant the native b subunit partners. Hence, substitu- M-Hbs have contributed greatly to understand- tion of the normal His F8 side chain for a longer ing the biochemical properties of the heme iron, Tyr F8 (Fig. 4A) also stabilizes the deoxygenated including its interactions with various ligands T state and reduces O2 affinity of the native b and nearby amino acids such as the proximal subunit in Hb M-Iwate (Nagai et al. 2000; Jin and distal histidines. et al. 2004). These biochemical and structural alterations underlie the lack of cooperativity Globin Chain Elongation Mutants and severe cyanosis in patients with Hb M- Iwate, in which metHb levels can exceed 20% Antitermination and frameshift mutations that (normal ,2%) (Ameri et al. 1999). In contrast, add irrelevant amino acids to the carboxyl ter- Hb M-Saskatoon (b63 [E7] His . Tyr) re- minus of globin proteins produce interesting places the distal His with Tyr (Fig. 4D) (Horlein variants that can damage erythrocytes (Nathan and Weber 1948; Hayashi et al. 1966). In this et al. 2009). The most clinically significant

ACB Iwate (α F8 Tyr) Normal

E7 metHb N N N N N N H H reductase H H O 2 system Fe3+ Fe3+ Fe2+

Tyr F8 O N N N N F8

D E Saskatoon (β E7 Tyr)

www.perspectivesinmedicine.org Tyr E7 E7 metHb H+ reductase – O H O system OH Fe3+ Fe3+ Fe2+

N N N N N N F8

Figure 4. Examples of M-type hemoglobins. (A) The heme pocket of wild-type Mb (gray) and the F8 His . Tyr mutation (orange, PDB 1HRM), which serves as a model for Hb M-Iwate. Effects on the protein fold are to increase 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. This ferric form is not reduced by met-Hb reductase. (C) With the normal His F8 present, ferric heme is readily reduced. (D) Substituting the distal His E7 side chain in Mb for a larger Tyr E7 (orange, PDB 1MGN), as also occurs 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 reduced by met-Hb reductase, possibly owing to a transient protonation of Tyr E7. Note that Mb is used as a model for Hb subunits in A and D, whereas B and E are based on Hb spectroscopy.

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Hemoglobin Variants

example is Hb Constant Spring (a2 142 [HC3] get et al. 1975). Cross-comparison of the protein Stop . Gln), caused byan antitermination mu- and mRNA sequencing data allowed Bunn, For- tation at the a2 stop codon (Clegg et al. 1971; get, and colleagues to more rapidly define nor- Efremov et al. 1971; Milner et al. 1971; Clegg mal b-globin gene structures and ascertain that and Weatherall 1974). This elongates the pro- the Hb Cranston mutation likely arose by non- tein by 31 amino acids, generating an unstable homologous crossover of two normal b-globin protein that is relatively underrepresented in genes. hemolysates, but can be detected by physical methods (see sections on Basic Principles, Lab- Variants that Affect Multiple Hemoglobin oratory Testing for Hb Variants, and Physical Functions methods of Hb separation). In addition, Hb Constant Spring mRNA is rapidly degraded in Not surprisingly, amino acid substitutions with- developing erythrocytes, owing to ribosomal in critical regions of globin proteins can pro- entry into the 30UTR, causing displacement of duce multiple effects. For example, HbE (b26 RNA-bound stabilizing proteins with a resul- [B8] Glu . Lys), a common variant in South- tant thalassemia syndrome (Hunt et al. 1982; east Asia, contains an amino acid substitution Derry et al. 1984; Weiss and Liebhaber 1994; that renders b chains mildly unstable in vitro Morales et al. 1997). with minimal clinical significance (Frischer Hb Constant Spring contributesto a-thalas- and Bowman 1975; Huisman 1997; Rees et al. semia syndromes, particularly when combined 1998; see also Musallam et al. 2012). However, with two a-globin deletional alleles (–/aCSa), this mutation also creates an alternate splice site which produces a severe form of HbH disease in the b-globin mRNA, leading to reduced syn- (Viprakasit and Tanphaichitr 2002). Isolated thesis of productive transcripts with resultant Hb Constant Spring, in its heterozygous (aa/ thalassemia (Orkin et al. 1982). HbE is particu- aCSa) or homozygous (aCSa/aCSa) forms, re- larly deleterious when coinherited with a more sults in more severe anemia than occurs when severe b-thalassemic allele, which happens com- the same a alleles are deleted (aa/2a)or monly in Southeast Asia. (2a/2a) (Schrier et al. 1997). This is due to Mutations that alter the heme pocket com- the cytotoxic effects of the unstable Constant monly produce multiple biochemical effects. Spring protein. Although most common in For example, deletion or substitution of the Southeast Asia, Hb Constant Spring is increas- conserved Phe residue at the CD1 helical region ingly identified in other geographic regions, in the heme pocket markedly destabilizes the largely through global migration (Lal et al. affected globin and also alters its O2 affinity. www.perspectivesinmedicine.org 2011). In fact, it was first discovered in a Chinese Thus, Hb Bruxelles (b42 [CD1] Phe . 0) family living in Constant Spring, Jamaica. (Blouquit et al. 1989; Griffon et al. 1996), Hb One example of a b-globin chain elongation Warsaw (b42 [CD1] Phe . Val) (Honig et al. mutant is Hb Cranston (b 145 [HC3] þCT) 1990), Hb Hammersmith (b42 [CD1] Phe . (Bunn et al. 1975). This mutation introduces a Ser) (Dacie et al. 1967), and Hb Buccuresti-Lou- frameshift at the normal stop codon to generate isville (b42 [CD1] Phe . Leu) (Bratu et al. a b chain that is extended by 11 amino acids. 1971; Keeling et al. 1971) cause both congenital This results in an unstable Hb tetramer with Heinz body hemolytic anemia and cyanosis. high O2 affinity and diminished cooperativity These combined effects arise from severely re- (McDonald et al. 1980; Shaeffer et al. 1980). Af- duced cooperativity, rapid rates of autoxidation fected patients show compensated hemolytic and hemin loss, and unfolding of these unstable anemia with the variants accounting for 30% globin variants (Griffon et al. 1996). of total Hb in the hemolysate. Interestingly, the Another interesting heme pocket variant is structure of Hb Cranston was investigated Hb Zurich (b63 [E7] His . Arg) in which the simultaneously with studies to determine the distal His is replaced by Arg (Huisman et al. b-globin mRNA 30 untranslated sequence (For- 1961; Bachmann and Martihr 1962; Frick et al.

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C.S. Thom et al.

1962; Tucker et al. 1978; Phillips et al. 1981; bon monoxide (CO) binding. As a result, indi- Springer et al. 1989). The large, highly polar viduals with Hb Zurich tend to have supranor- variant His side chain swings out of the distal mal levels of CO-Hb, which ironically protects heme pocket, and the positively charged guani- the heme iron from oxidation and the globin dino group forms a salt bridge with a heme from denaturation. Affected subjects who are propionate (Fig. 5A). This results in an enlarged cigarette smokers accumulate even higher levels ligand-binding pocket, destabilizing O2 binding of CO-Hb, which tendsto protect against hemo- and causing iron autoxidation via exposure to lysis. Thus, “the pathologyof a mutant protein is water. Affected individuals show increased sen- ameliorated by a normally toxic pollutant” sitivity to oxidant agents, including sulfa drugs, (Bunn and Forget 1986). which more easily enter the expanded heme Two other recently identified Hb variants pocket. Loss of the distal histidine markedly illustrate how multiple biochemical defects decreases O2 affinity but has little effect on car- produce unique phenotypes. Hb Jamaica Plain

A Phe CD4 Phe CD4

Phe CD1 Phe CD1

Arg E7 Arg E7 His E7 His E7 His F8 His F8

B

His E7 His E7

Phe CD1 Phe CD1

www.perspectivesinmedicine.org Trp E11 Trp E11 Thr E11 Thr E11 His F8 His F8 Val E11 Val E11

Figure 5. Hb variants with amino acid changes in the heme pocket. (A) Stereo diagram of a model of the deoxy Hb Zurich b heme pocket (blue) overlaid with the wild-type b heme pocket (black, PDB 2DN2). This model of Hb Zurich is based on the structures solved by Phillips et al. (1981) and Tucker et al. (1978). It was generated with the macromolecular modeling program Coot (Emsley et al. 2010) by mutating the distal His of deoxy-b (PDB 2DN2) to Arg. The distal Arg E7 (red) is oriented toward the CD corner, disturbing the position of Phe CD1 and Phe 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 diagram showing the structural changes associated with substitution of b Val E11. The wild-type structure carrying the branched hydrophobic side chain Val (black bonds, PDB 2DN2) is overlaid with structures carrying the largest aromatic side chain Trp E11 (orange, PDB 101K) or a polar side chain Thr E11 (green PDB, 1HDB). It is clear that no major backbone or side chain repacking occurs. Thus, mutations in this position are likely to have specific effects in changing the volume of the heme pocket accessible to solvent or diatomic ligands and the electrostatic properties of the pocket. These changes will manifest as differences in O2 binding, ligand selectivity, autoxidation, and heme loss.

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Hemoglobin Variants

(b6[A3]Glu. Val and b68 [E12] Leu . Phe) heme pocket. However, subsequent DNA anal- contains two defects in the same b chain, a b6 ysis of the same patient identified a Val . Met Glu to Val substitution that causes sickle cell codon substitution (Rees et al. 1996). The inves- anemia (Serjeant and Rodgers 2012) and a b68 tigators concluded that the mutant Met residue amino acid substitution that reduces O2 affini- was converted to Asp posttranslationally, prob- ty, probably by destabilizing the oxygenated ably through oxidative mechanisms. More re- conformation through steric effects introduced cently, the analogous variant was identified in into helix E (Geva et al. 2004). The affected pa- fetal (g) globin (Hb Toms River) (Crowley tient, who was heterozygous for the mutant al- et al. 2011). The affected patient was a newborn lele, showed symptoms of sickle cell anemia that presenting with both cyanosis and anemia. DNA were precipitated by infection or airplane travel. testing revealed the codon change (Val . Met at Thus, an amino acid substitution that reduces E11). Mass spectrometry of patient hemolysate O2 affinity exacerbates the effects of a sickling indicated a mixture of variant g globins contain- mutation in the same globin chain. ing either Met or Asp at position E11. Although Several other Hb variants modulate the se- no structural studies have been performed with verity of sickle cell anemia (see also Cao and Kan Hb chains carrying Met or Asp E11, structures 2012; Schechter and Elion 2012; Serjeant and with polar (Thr) or large aromatic (Trp) substi- Rodgers 2012). For example, g globin inhibits tutions are available. These indicate that a range polymerization of HbS (Nagel et al. 1979). This of amino acids can be accommodated without effect is attributable to differences in several gross changes in the heme pocket structure (Fig. amino acid residues compared with the corre- 5B). Instead, altered steric and electrostatic in- sponding b chain, including g80 and g87 (Ada- teractions with the distal His and diatomic li- chi and Asakura 1979; Nagel et al. 1979; Adachi gands entering the heme pocket are likely to be et al. 1996). Hb D-Ibadan (b87 [F3] Thr . Lys), functionally significant. Biochemical studies in- which introduces a lysine residue at the b87 po- dicated that the Hb TomsRiver Met substitution sition, is presumed to have decreased interaction produced a stable, low O2 affinity variant g glo- with the mutant Valresidue at HbS b6 (Watson- bin, causing cyanosis. Its gradual posttransla- Williams et al. 1965; Nagel et al. 1979). Thus, Hb tional conversion to Asp destabilized the mole- D-Ibadan inhibits HbS polymerization. In con- cule, causing hemolytic anemia. This provides trast, Hb Quebec-Chori (b87 [F3] Thr . Ile) an example of how posttranslational modifica- was identified in a compound heterozygous pa- tions of variant globins can modify phenotypes. tient with mild to moderately severe sickle cell The reason that Hb Bristol-Alesha causes pre- anemia (Witkowska et al. 1991). The introduc- dominantly hemolytic anemia whereas Hb www.perspectivesinmedicine.org tion of an isoleucine at b87 causes Hb Quebec- Toms River causes mainly cyanosis probably re- Chori to promote deoxygenated HbS polymer- flects different rates of Met conversion to Asp in ization. the variant globin chains. Hb Bristol-Alesha (b67 [E11] Val . Met) (Molchanova et al. 1993; Rees et al. 1996) and CONCLUDING REMARKS Hb Toms River (g67 [E11] Val . Met) (Crow- ley et al. 2011), which contain analogous amino More than 1000 Hb variants are known to exist acid substitutionsin b and g chains, respectively, (Globin Gene Server; Hardison et al. 2002; Giar- represent interesting globin variants with mul- dine et al. 2011). These are mainly missense tiple biochemical defects. Hb Bristol-Aleshawas mutations that destabilize Hb, alter Hb-O2 af- initially observed in patients with moderately finity, or most commonly, alter Hb function severe hemolytic anemia. Studies of the mutant minimally. Moreover, variants that do alter Hb protein in patient erythrocytes revealed a b67 biochemistry are rarely life threatening or health Val . Asp substitution, predicted to render compromising. Nonetheless, studies of these the protein unstable by introducing a highly variants have been of great benefit to science charged polar residue into the hydrophobic and medicine for two main reasons. First,

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identification of Hb gene mutations as a cause Arous N, Braconnier F, Thillet J, Blouquit Y, Galacteros F, for cyanosis, erythrocytosis, or mild hemolysis Chevrier M, Bordahandy C, Rosa J. 1981. Hemoglobin Saint Mande´ [b102 (G4) Asn!Tyr]: A new low oxygen in otherwise healthy patients provides reassur- affinity variant. FEBS Lett 126: 114–116. ance and minimizes additional diagnostic pro- Asakura T, Adachi K, Shapiro M, Friedman S, Schwartz E. cedures, sparing expense and risk. Second, ef- 1975. Mechanical precipitation of hemoglobin ko¨ln. Bio- forts to understand how Hb variants produce chim Biophys Acta 412: 197–201. their structural, biochemical, and clinical effects Bachmann F,Marti HR. 1962. Hemoglobin Zu¨rich. II. Phys- icochemical properties of the abnormal hemoglobin. has generated important insights into red blood Blood 20: 272–86. cell function and also created general paradigms Bare GH, Bromberg PA, Alben JO, Brimhall B, Jones RT, for the study of protein biology. Despite hun- Mintz S, Rother I. 1976. Altered C-terminal salt bridges dreds of studies over more than 50 years, new Hb in haemoglobin York cause high oxygen affinity. Nature 259: 155–156. variants continue to emerge, yielding new in- Bender JW,Adachi K, Asakura T.1981. Precipitation of oxy- sights into this important molecule. hemoglobins A and S by isopropanol. Hemoglobin 5: 463–474. Blouquit Y, Bardakdjian J, Lena-Russo D, Arous N, ACKNOWLEDGMENTS Perrimond H, Orsini A, Rosa J, Galacteros F. 1989. Hb Bruxelles: a 2A b (2)41 or 42(C7 or CD1)Phe deleted. Wethank Drs. Franklin Bunn, Kazuhiko Adachi, Hemoglobin 13: 465–474. and John Olson for comments on the manu- Bonaventura J, Riggs A. 1968. Hemoglobin Kansas, a human script. Hemoglobin research in Dr. Weiss’s lab- hemoglobin with a neutral amino acid substitution and an abnormal oxygen equilibrium. J. Biol Chem 243: 980– oratory is funded through National Institutes of 991. Health (NIH) grants DK061692, HL087427, Bonaventura J, Bonaventura C, Sullivan B, Ferruzzi G, and P30DK090969. The authors declare no McCurdy PR, Fox J, Moo-Penn WF. 1976. Hemoglobin competing financial interests. providence. Functional consequences of two alterations of the 2,3-diphosphoglycerate binding site at position b82. J. Biol Chem 251: 7563–7571. Bratu V,Lorkin PA, Lehmann H, Predescu C. 1971. Haemo- REFERENCES globin Buccures¸ti 42(CD1) Phe-Leu, a cause of unstable Reference is also in this collection. haemoglobin haemolytic anaemia. Biochim Biophys Acta 251: 1–6. Adachi K, Asakura T.1979. The solubility of sickle and non- Bunn HF, Forget BG. 1986. Hemoglobin: Molecular, genetic sickle hemoglobins in concentrated phosphate buffer. and clinical aspects. W.B. Saunders, Philadelphia. J. Biol Chem 254: 4079–4084. Bunn HF, Wohl RC, Bradley TB, Cooley M, Gibson QH. Adachi K, Pang J, Konitzer P,Surrey S. 1996. Polymerization 1974. Functional properties of hemoglobin Kempsey. J of recombinant hemoglobin F gE6V and hemoglobin F Biol Chem 249: 7402–7409. gE6V, gQ87Talone, and in mixtures with hemoglobin S. Bunn HF, Schmidt GJ, Haney DN, Dluhy RG. 1975. Hemo- Blood 87: 1617–1624.

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Hemoglobin Variants: Biochemical Properties and Clinical Correlates

Christopher S. Thom, Claire F. Dickson, David A. Gell and Mitchell J. Weiss

Cold Spring Harb Perspect Med published online February 6, 2013

Subject Collection Hemoglobin and Its Diseases

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

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

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