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Classification of the Disorders of Hemoglobin

Bernard G. Forget and H. Franklin Bunn

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

Subject Collection Hemoglobin and Its Diseases

Hemoglobin Variants: Biochemical Properties and The Prevention of Thalassemia

Clinical Correlates Antonio Cao and Yuet Wai Kan
Christopher S. Thom, Claire F. Dickson, David A.
Gell, et al.
Classification of the Disorders of Hemoglobin The Switch from Fetal to Adult Hemoglobin
Bernard G. Forget and H. Franklin Bunn Vijay G. Sankaran and Stuart H. Orkin
The Molecular Basis of -Thalassemia Pathophysiology and Clinical Manifestations of
Douglas R. Higgs the -Thalassemias
Arthur W. Nienhuis and David G. Nathan
Evolution of Hemoglobin and Its Genes Development of Gene Therapy for Thalassemia
Ross C. Hardison Arthur W. Nienhuis and Derek A. Persons
The Search for Genetic Modifiers of Disease -Thalassemia, Mental Retardation, and
Severity in the -Hemoglobinopathies Myelodysplastic Syndrome
Guillaume Lettre Richard J. Gibbons
World Distribution, Population Genetics, and -Thalassemia Intermedia: A Clinical Perspective
Health Burden of the Hemoglobinopathies Khaled M. Musallam, Ali T. Taher and Eliezer A.
Thomas N. Williams and David J. Weatherall Rachmilewitz
Iron Metabolism: Interactions with Normal and Hematopoietic Stem Cell Transplantation in
Disordered Erythropoiesis Thalassemia and Sickle Cell Anemia
Tomas Ganz and Elizabeta Nemeth Guido Lucarelli, Antonella Isgr, Pietro Sodani, et
Pluripotent Stem Cells in Research and Treatment
of Hemoglobinopathies
Natasha Arora and George Q. Daley

For additional articles in this collection, see

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Classification of the Disorders

of Hemoglobin

Bernard G. Forget1 and H. Franklin Bunn2

Section of Hematology, Department of Medicine, Yale School of Medicine, New Haven,
Connecticut 06520-8028
Hematology Division, Department of Medicine, Brigham and Womens Hospital, Harvard Medical
School, Boston, Massachusetts 02115

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

ture and function of hemoglobin, along with the mechanism of assembly of its subunits,
as background for the rationale and basis of the different categories of disorders in the

n impressive degree of molecular engineer- tion to Fe3, which is incapable of binding ox-
A ing was necessary for the evolution of a
multisubunit protein that higher organisms re-
ygen.3 Delicate noncovalent interactions be-
tween unlike globin subunits are required for
quire for optimal oxygen homeostasis and buf- the hemoglobin tetramer a2b2 to bind and un-
fering of acidic metabolic waste products. Each load oxygen in a cooperative manner, thereby
globin subunit must form a stable linkage with assuring maximal transport to actively metab-
heme (ferroprotoporphyrin IX) situated on the olizing cells. This phenomenon is reflected by
external surface of the protein so that oxygen a sigmoid oxygen-binding curve that depends
in the RBC cytosol can bind reversibly to the on hemoglobin tetramer having two quaternary
hemes iron atoms. Moreover, the hydrophobic structures: the T or deoxy conformer that has
cleft into which the heme is inserted must be low oxygen affinity and the R or oxy conformer
able to protect the Fe2 heme iron from oxida- that has high oxygen affinity. Further fine-

For more detailed information, see Dailey and Meissner (2013).
Editors: David Weatherall, Alan N. Schechter, and David G. Nathan
Additional Perspectives on Hemoglobin and Its Diseases available at
Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011684
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B.G. Forget and H.F. Bunn

tuning of hemoglobin function comes from its mutations of globin genes that impair synthesis
allosteric behavior triggered by the binding of give rise to thalassemia and anemia of varying
two small effector molecules 2,3-BPG, and pro- degree. In addition, well-defined clinical and
tons to specific sites on the T structure distant hematologic phenotypes are associated with
from the heme groups.4 To endow the blood mutations that alter the structure of globin sub-
with high oxygen carrying capacity hemoglo- units, discussed in more detail in Thom et al.
bin must be stuffed into flexible circulating (2013). Impairment of hemoglobin solubility
RBCs. A remarkably high degree of solubility can be caused either by the formation of intra-
is required for hemoglobin to achieve an intra- cellular polymers (sickle cell disease) or by the
cellular concentration of 34 g/dl or 5 mM development of amorphous precipitates (con-
(tetramer). genital Heinz body hemolytic anemia). Abnor-
To reach such a high corpuscular hemoglo- malities of oxygen binding can lead either to
bin concentration it is essential that a-globin as erythrocytosis (high O2 affinity mutants) or to
well as b-globin (or g-globin) mRNA be ex- cyanosis (low O2 affinity mutants). Some glo-
pressed at very high levels during erythroid dif- bin mutants have structural alterations within
ferentiation. Moreover, a- and non-a-globin the heme pocket that result in oxidation of
synthesis must be closely matched. As explained the heme iron and pseudocyanosis because of
in Nienhuis and Nathan (2012), subunit imbal- methemoglobinemia.
ance is central to the pathophysiologyof the thal-
assemias. Free a-globin subunits are particularly
toxic to erythroid cells. This threat is alleviated GENERAL CLASSIFICATION OF
by the presence of a-hemoglobin stabilizing HEMOGLOBIN DISORDERS
protein (AHSP), a chaperone that is expressed

Hemoglobin disorders can be broadly classified

at high levels in erythroid cells and binds specif-
into two general categories (as listed in Table 1):
ically and tightly to heme-intact a-globin sub-
units (Kihm et al. 2002; Feng et al. 2004; Mollan 1. Those in which there is a quantitative defect
et al. 2010, 2012). The AHSP protects the cell in the production of one of the globin sub-
from potentially toxic oxidized (Fe3) heme un- units, either total absence or marked reduc-
til it is reduced to functional Fe2 heme by cy- tion. These are called the thalassemia syn-
tochrome b5 reductase. On encountering a free dromes.
heme-intact b-globin subunit, the a-globin
2. Those in which there is a structural defect in
dissociates from AHSP to form the extremely
one of the globin subunits.
stable ab dimer. As discussed later in this
work, this process is facilitated by electrostatic The majority of human hemoglobin mu-
attraction between positively charged a-globin tants were discovered as an incidental finding,
subunits and negatively charged b-globin sub- unassociated with any hematologic or clinical
units. phenotype. However, a number of a- and b-glo-
In view of the multiple molecular con- bin mutants are associated with distinct clinical
straints that are required for high-level produc- phenotypes. These fall into five broad catego-
tion of fully functional and highly soluble he- ries: the sickle syndromes (SS, SC, Sb0-thalasse-
moglobin, it is no surprise that Murphys law mia, and Sb-thalassemia); unstable mutants
is in full force: whatever can go wrong will. causing congenital Heinz body hemolytic ane-
As discussed briefly here, and in much more mia; mutants with high oxygen affinity result-
detail in Thein (2013), Higgs (2013), Nienhuis ing in erythrocytosis; low oxygen affinity mu-
and Nathan (2012), and Musallam et al. (2012), tants and the M hemoglobins causing cyanosis;
and mutants associated with a thalassemia phe-
notype. Most of these disorders are inherited
For more detailed information on hemoglobin function, genetic defects, but there are some defects that
see Schechter (2013). are acquired or occur de novo.

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Classification of Hemoglobin Disorders

Table 1. Classification of hemoglobin disorders

The thalassemia syndromes HEMOGLOBIN
A. b-Thalassemia A. Sickle cell disorders
Clinical classification: SA, sickle cell trait
b-Thalassemia minor or trait SS, sickle cell anemia/disease
b-Thalassemia major SC, HbSC disease
b-Thalassemia intermedia S/b thal, sickle b-thalassemia disease
Biochemical/genetic classification: S with other Hb variants: D, O-Arab, other
b0-Thalassemia SF, Hb S/HPFH
b-Thalassemia B. Hemoglobins with decreased stability
d-Thalassemia (unstable hemoglobin variants)
g-Thalassemia Mutants causing congenital Heinz body hemolytic
Lepore fusion gene anemia
db-Thalassemia Acquired instabilityoxidant hemolysis: Drug-
1gdb-Thalassemia induced, G6PD deficiency
C. Hemoglobins with altered oxygen affinity
Dominant b-thalassemia (structural variants with
High/increased oxygen affinity states:
b-thalassemia phenotype)
Fetal red cells
b-Thalassemia with other variants: Decreased RBC 2,3-BPG
HbS/b-thalassemia Carboxyhemoglobinemia, HbCO
HbE/b-thalassemia Structural variants

Low/decreased oxygen affinity states:

B. a-Thalassemia Increased RBC 2,3-BPG
Deletions of a-globin genes: Structural variants
One gene: a-thalassemia
Two genes in cis: a0-thalassemia D. Methemoglobinemia
Two genes in trans: homozygous a-thalassemia Congenital methemoglobinemia:
( phenotype of a0-thalassemia) Structural variants
Three genes: HbH disease Cytochrome b5 reductase deficiency
Four genes: Hydrops fetalis with Hb Barts Acquired (toxic) methemoglobinemia
Nondeletion mutants: E. Posttranslational modifications
Hb Constant Spring Nonenzymatic glycosylation
Other Amino-terminal acetylation
C. De novo and acquired a-thalassemia Amino-terminal carbamylation
a-Thalassemia with mental retardation syndrome (ATR): Deamidation
Due to large deletions on chromosome 16 involving
the a-globin genes
Due to mutations of the ATRX transcription factor
gene on chromosome X
a-Thalassemia associated with myelodysplastic
syndromes (ATMDS):
Due to mutations of the ATRX gene

THE THALASSEMIA SYNDROMES subunits of hemoglobin. In the alpha (a)-thal-

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

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B.G. Forget and H.F. Bunn

thalassemias affecting the production of delta (b-thalassemia major), there is usually a severe
(d)- or gamma (g)-globin subunits have also transfusion-dependent hemolytic anemia asso-
been described but are not clinically signif- ciated with marked ineffective erythropoiesis
icant disorders. Combined deficiency of d resulting in destruction of erythroid precursor
b-globin subunits, or of all of the b-like glo- cells in the bone marrow.
bin subunits also occurs. These disorders are A less common clinical phenotype is re-
described in detail in Thein (2013) and Higgs ferred to as b-thalassemia intermedia. In this
(2013). clinical disorder, there is a moderate to severe,
partially compensated, hemolytic anemia that
does not require chronic transfusion therapy
THE b-THALASSEMIAS to maintain a satisfactory circulating hemo-
globin level in the affected patient, although
The b-thalassemias can be subclassified into occasional transfusions may be required if the
those in which there is total absence of normal anemia worsens because of associated com-
b-globin subunit synthesis or accumulation, plications. Such patients have a milder disease
the b0-thalassemias, and those in which some because there is less severe a- to non-a-globin
structurally normal b-globin subunits are syn- subunit imbalance than in typical b-thalasse-
thesized, but in markedly decreased amounts, mia major patients, resulting in less accumu-
the b-thalassemias. The molecular basis of the lation of free a-subunits that cause the ineffec-
b-thalassemias is very heterogeneous (see Thein tive erythropoiesis. There are different possible
2013), with over 200 different mutations having causes for such a lessened a- to non-a-globin
been described. In general, the mutations caus- subunit imbalance, including: inheritance of
ing b-thalassemia are point mutations affect- milder b-thalassemia mutations with less se-

ing a single nucleotide, or a small number of vere than usual deficiency of b-globin subunit
nucleotides, in the b-globin gene. Rare deletion production; coinheritance of a form of a-thal-
forms of b-thalassemia have also been de- assemia; or coinheritance of another genetic
scribed. One of these deletions is caused by un- trait associated with increased production of
equal crossing over between the linked and the g-subunit of fetal hemoglobin. Most pa-
partially homologous d- and b-globin genes, tients with b-thalassemia intermedia carry
resulting in the formation of a fusion db-globin two mutant b-globin genes: they have a geno-
gene, the Lepore gene, that has a low level of type typical of b-thalasemia major, but the phe-
expression. Large deletions involving part or notype is modified by one of the factors listed
all of the b-globin gene cluster are responsible above. However, rare cases of b-thalassemia in-
for the db-thalassemias, the 1gdb-thalassemias, termedia are caused by heterozygosity for a
and the hereditary persistence of fetal hemoglo- single mutant b-globin gene associated with
bin (HPFH) syndromes. the production of a highly unstable b-globin
Despite the marked heterogeneity in the subunit that causes RBC damage in a fashion
molecular basis of the b-thalassemias, the clin- similar to excess free a-subunits; this is the
ical phenotype of these disorders is relatively so-called dominant b-thalassemia (Thom et
homogeneous because of their common patho- al. 2013).
physiology: deficiency of HbA tetramers and The db-thalassemias are associated with to-
excess accumulation of free a-subunits incapa- tal deficiency of b-globin subunit production,
ble of forming hemoglobin tetramers because of but are clinically milder than typical cases of b0-
deficiency of b-like globin subunits (see Nien- thalassemia, because there is an associated per-
huis and Nathan 2012). In heterozygotes (b- sistent high level of expression of the g-subunit
thalassemia trait or b-thalassemia minor), there of fetal hemoglobin that decreases the degree
is mild to moderate hypochromic microcytic of a-subunit excess. The 1gdb-thalassemias are
anemia, without evidence of hemolysis, whereas associated with neonatal hemolytic anemia,
in homozygotes or compound heterozygotes but this resolves during the first few months

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Classification of Hemoglobin Disorders

of life and the associated phenotype in adults is treatment by RBC transfusion. The basis of
that of b-thalassemia trait or b-thalassemia mi- the hemolysis is the excess accumulation of b-
nor. The syndrome of hereditary persistence of globin subunits that self-associate to form
fetal hemoglobin (HPFH) is not, strictly speak- b-chain tetramers or HbH. In contrast to the
ing, a form of b-thalassemia because it is not situation in the b-thalssemias in which excess
associated with significant a- to non-a-globin a-globin subunits rapidly form insoluble ag-
subunit imbalance, but is characterized by high gregates, excess b-globin chains can form sol-
levels of persistent g-globin production and is uble tetramers (HbH). However, HbH is rela-
frequently considered within the spectrum of tively unstable and does precipitate as RBCs age,
db-thalassemia. forming inclusion bodies that damage RBCs
and shorten their lifespan (see Higgs 2013).
The deletion of all four a-globin genes is usually
THE a-THALASSEMIAS fatal during late pregnancy or shortly after birth.
This condition is called hydrops fetalis with Hb
In contrast to the b-thalassemias, which are Barts. Hb Barts is a tetramer of four g-globin
usually caused by point mutations of the b-glo- subunits and is ineffective as an oxygen trans-
bin gene, the a-thalassemia syndromes are usu- porter: it has a very high oxygen affinity, similar
ally caused by the deletion of one or more a- to that of myoglobin, and does not release ox-
globin genes and are subclassified according to ygen to the tissues under physiologic condi-
the number of a-globin genes that are deleted tions. Therefore, the infant, whose RBCs lack
(or mutated): one gene deleted (a-thalasse- HbF or HbA and contain mostly Hb Barts, suf-
mia); two genes deleted on the same chromo- fers severe hypoxia resulting in hydrops fetalis.
some or in cis (a0-thalassemia); three genes Rare cases have been rescued by intrauterine

deleted (HbH disease); or four genes deleted transfusions, but these children subsequently
(hydrops fetalis with Hb Barts). Nondeletion require lifelong transfusion support similar to
forms of a-thalassemia have also been charac- that required by children with b-thalassemia
terized but are relatively uncommon. These dis- major.
orders are discussed in detail in Higgs (2013). One form of nondeletion a-thalassemia,
Clinically, the deletion of only one of the called Hb Constant Spring (HbCS), is particu-
four a-globin genes is not associated with larly prevalent in southeast Asia. It is caused by a
significant hematologic abnormalities and is chain termination mutation, which results in the
sometimes called the silent carrier state for synthesis of an elongated a-globin subunit that
a-thalassemia. The deletion of two a-globin accumulates at very low levels in RBCs of affect-
genes can occur in two forms: (1) on the same ed individuals. When coinherited with the a0-
chromosomes, or in cis; or (2) on opposite chro- cis deletion on the other chromosome, there re-
mosomes, or in trans, which is the homozygous sults a form of HbH disease that is more severe
state for the single gene deletion, or homozygous than the typical HbH disease associated with
a-thalassemia. The cis genotype is particular- full deletion of three a-globin genes (see Higgs
ly common in Asian populations, whereas the 2013).
trans genotype is highly prevalent in persons of In addition to these forms of a-thalassemia
black/African ancestry. The clinical phenotype inherited in a pattern consistent with Mendelian
is similar with both genotypes and consists genetics, there are two a-thalassemia syndromes
of mild hypochromic, microcytic anemia, with- that are caused by de novo or acquired muta-
out hemolysis, somewhat analogous to that of tions affecting expression of the a-globin genes:
b-thalasssemia trait, but somewhat less severe. (1) the a-thalassemia with mental retardation
The deletion (or markedly decreased expression) syndrome (ATR); and (2) acquired a-thalasse-
of three a-globin genes is associated with the mia (HbH disease) associated with myelodys-
syndrome of HbH disease, a compensated he- plastic syndromes (ATMDS). These syndromes
molytic anemia that usually does not require are described in detail in Gibbons (2012).

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B.G. Forget and H.F. Bunn

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

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

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Classification of Hemoglobin Disorders

and concentration of HbS in the red cell. The does not participate at all in polymer forma-
kinetics of sickling are markedly concentration tion, this is sufficient to prevent sickling of all
dependent, the speed of the reaction being in- RBCs under physiologic conditions. This situa-
versely proportional to about the 30th power of tion is in contrast to what occurs when variable
the starting HbS concentration. Thus, a small amounts of HbF are present in HbSS disease or
increase in intracellular HbS concentration, the sickle/b-thalassemia syndromes. In the lat-
such as that resulting from loss of intracellular ter disorders, the HbF is distributed in a hetero-
water and cellular dehydration, can have a major cellular fashion, in which the HbF is present
effect on the speed of the polymerization reac- in only a subset of cells called F cells, leaving
tion. The amount and type of other non-S he- the other cells that lack HbF unprotected from
moglobins in the red cell can also influence the the antisickling effects of HbF. Nevertheless, a
extent or rate of sickling, and thus clinical se- marked increase in the proportion of F cells in
verity. HbSC disease is associated with signifi- such cases can have a beneficial effect on the
cant clinical manifestations. Why do SC pa- clinical manifestation of these sickling disor-
tients often have vaso-occlusive manifestations ders. Therefore, there is a great deal of interest
as dramatic as those with SS disease, whereas and ongoing research to develop strategies for
AS individuals have virtually no morbidity? enhancing HbF synthesis and F-cell production
HbC and HbA copolymerize to an identical de- as a therapeutic modality for sickling disorders
gree with HbS (Bunn et al. 1982). Two indepen- (see Sankaran and Orkin 2013).
dent factors conspire to make HbSC a disease.
The presence of HbC in the red cell greatly en-
hances potassium efflux and red cell dehydra-
tion, which increases corpuscular hemoglobin A substantial minority of Hb mutants have sub-

concentration (Bunn et al. 1982) and promotes stitutions that alter the solubility of the molecule
polymerization. Sickling in SC patients is fur- in the red cell. The intraerythrocytic precipitated
ther enhanced by the fact that they have 50% material derived from the unstable abnormal Hb
HbS, whereas HbAS individuals have 40% is detectable by a supravital stain as dark globu-
HbS. This difference is because a-globin sub- lar aggregates called Heinz bodies (HBs). These
units bind more readily to negatively charged intracellular inclusions reduce the life span of
bA-subunits than to positively charged bC-sub- the red cell and generate a hemolytic process of
units, as explained further at the end of this varied severity called congenital Heinz body he-
work. Other clinically significant sickle cell syn- molytic anemia. When a red cell hemolysate of
dromes include: sickle/b0-thalassemia, sickle/ an affected individual is heated to 508C or treat-
b-thalassemia, HbSD disease, and HbSO- ed with 17% isopropanol, a precipitate usually
Arab disease. HbD and HbO-Arab participate develops. The hemoglobin electrophoresis often
more readily than HbA in copolymer formation reveals an abnormal banding pattern. Defini-
with HbS. tive diagnosis requires analysis of either globin
A striking example of how another hemo- structure or DNA sequence. For more informa-
globin can dramatically influence sickling is the tion on congenital Heinz body hemolytic ane-
condition that results from coinheritance of mia and other hemoglobin variants summa-
HbS with hereditary persistence of fetal hemo- rized in this section, see Thom et al. (2013).
globin: HbSF or S/HFPH. This condition, in
which there is 70% HbS and 30% HbF,
is clinically asymptomatic and is a notable ex-
ception to the general rule that clinical severity
is directly related to the amount (or %) of HbS Over 25 hemoglobin variants have been en-
in the RBC. In this condition, the HbF is dis- countered in individuals with erythrocytosis.
tributed in a pancellular fashion so that every Amino acid substitutions cause an increase in
RBC contains 30% HbF and, because HbF oxygen affinity usually because of impairment

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B.G. Forget and H.F. Bunn

in the stability of the T or deoxy quaternary it is stabilized as Fe3. Thus, the M hemoglobins
structure. Erythrocytosis is usually familial and include a58 His ! Tyr, a87 His ! Tyr, b63
is the only well-documented clinical finding. His ! Tyr, b92 His ! Tyr, g63 His ! Tyr,
Increased oxygen affinity results in a decrease and g92 His ! Tyr. In addition, b67 Val !
in hemoglobins ability to release oxygen during Glu results in very similar biochemical features
blood flow in the microcirculation. Unlike in- and clinical presentation. Affected individuals
dividuals with secondary erythrocytosis caused have cyanosis similar in hue to those with meth-
by high altitude exposure or to cardiac or pul- emoglobinemia in which the heme iron in nor-
monary hypoxemia, those having hemoglobin mal hemoglobin has been oxidized to Fe3.
with high oxygen affinity have normal arterial They have either normal hemoglobin levels
oxygen tension. Therefore, they are not hypox- or mild anemia and no clinical manifestations
emic. However, the reduction in unloading of other than their skin color.
oxygen to tissues results in cellular hypoxia.
Thus, the cells in the kidney that produce eryth-
ropoietin sense hypoxia and up-regulate Epo IMPACT OF SUBUNIT ASSEMBLY ON
gene expression leading to expansion of the HEMOGLOBIN PHENOTYPE
red cell mass. About half of the high-affinity The composition of the normal and mutant he-
variants can be detected by hemoglobin electro- moglobins in red cells of heterozygotes provides
phoresis. Definitive diagnosis is established by insight into the assembly of human hemoglo-
demonstration of a shift to the left in the oxy- bins (Bunn and McDonald 1983). The great ma-
hemoglobin binding curve. jority of b-subunit mutants are synthesized at
Less often, individuals and their relatives the same rate as bA and have normal stability.
will present with low O2 saturation and cyanosis

Therefore, heterozygotes would be expected to

owing to a hemoglobin variant with a marked have equal amounts of normal and mutant he-
reduction in oxygen affinity. The variants that moglobin. However, measurements of the pro-
have been most thoroughly characterized entail portion of normal and abnormal hemoglo-
amino acid replacements that destabilize the R bins in heterezygotes have revealed unexpected
or oxy quarternary structure. Affected indi- variability. Figure 1 shows a comparison of 105
viduals have normal hemoglobin levels and no stable b-subunit mutants. Positively charged
clinical manifestations other than the dusky mutants, such as hemoglobins S, C, D-Los An-
skin color. geles, and E5, constitute significantly less than
half of the total hemoglobin in heterozygotes
THE M HEMOGLOBINS and are reduced further in the presence of a-
thalassemia, because of increased competition
Rare families have attracted medical attention between normal and mutant b-subunits for lim-
because affected individuals have cyanosis ow- iting amounts of a-subunits. In contrast, many
ing to the inheritance of a hemoglobin variant of the negatively charged mutants are present in
in which an amino acid substitution within the amounts exceeding that of HbA. In two hetero-
heme pocket results in stabilization of the heme zygotes who had a negatively charged mutant
iron in the oxidized Fe3 form. As discussed in (HbJ-Baltimore or HbJ-Iran) in conjunction
Schechter (2013), among the most highly con- with a-thalassemia, the proportion of the mu-
served amino acids within hemoglobin sub- tant hemoglobin was further increased because
units are two histidines, one at helix position these bJ-subunits out-compete normal bA-sub-
F8, which binds to the heme iron and one at units for limiting amounts of a-subunits.
E7 in the pocket where oxygen and other ligands
bind to heme. Replacement of either of these
histidines by tyrosine in either the a-, b-, or Levels of HbE in AE heterozygotes are lower than the
others because the synthesis of bE-globin is impaired owing
g-globin subunit results in an alteration in the to a defect in mRNA splicing (see Fucharoen and Weatherall
electronic environment of the heme iron so that 2012 and Thein 2013).

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Classification of Hemoglobin Disorders

A 70 B



% of total hemoglobin
50 J-Iran

40 D-Los Ang

30 E

20 S-Oman

2 1 0 1 2 / / / /

Figure 1. Impact of surface charge on hemoglobin assembly. (A) Effect of charge on the proportion of abnormal
hemoglobin in individuals heterozygous for 105 stable b-globin variants. Each data point represents a mean
value for a given variant. Substitutions involving a histidine residue were scored as a change of one half charge.
The 21 group differs significantly from the 1 group (P , 0.001). (B) Effect of a-thalassemia on the

proportion of seven positively charged b-subunit variants (blue) and three negatively charged variants (red).
(Updated from Bunn and McDonald 1983 and Bunn 1987.)

Analyses of the proportion of b-subunit mutant Accordingly, in red cells of heterozygotes, it is

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

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B.G. Forget and H.F. Bunn

POSTTRANSLATIONAL ALTERATIONS acetylated at the amino terminus. These post-

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


HCOH HCOH Deamidation
NH2 +
HCOH 1 HCOH 2 HCOH In many proteins, the amino group on certain
HCOH HCOH HCOH asparagine residues, particularly when adjacent
CH2OH CH2OH CH2OH to a histidine, dissociates leaving aspartic acid.
Glucose Aldimine Ketoamine Deamidation does not occur on asparagines in
normal globin subunits. However, seven human
Reaction 1 shows the formation of the Schiff variants have been identified in which partial or
base or aldimine adduct. This product then re- complete deamidation occurs. Four of these en-
arranges to the more stable (virtually irrever- tail replacements by asparagine. The deamida-
sible) ketoamine linkage (reaction 2). HbAIc tion does not appear to impact function in any
and other less abundant glucose adducts are of these variants.
formed in a linear manner over the 120-day life
span of the red cell. Therefore, HbAIc levels are
low in individuals with hemolysis. Because the ACQUIRED ALTERATIONS IN
level of HbAIc is an accurate reflection of the HEMOGLOBIN FUNCTION
average concentration of plasma glucose over
the preceding 2 months, it has proven to be
very useful in monitoring therapeutic control Certain drugs and toxins can cause enhanced
in patients with diabetes. oxidation of heme iron to form methemo-
globin. Above levels of 10%, methemoglobin
individuals appear cyanotic. The extent of he-
Amino-Terminal Acetylation
moglobin oxidation is determined by spectro-
Shortly following translation of most proteins, photometric analysis of the blood. Methemo-
the amino-terminal (initiator) methionine is globinemia impairs oxygenation of tissues
cleaved and often the adjacent amino acid is because the Fe3 heme is incapable of binding

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Classification of Hemoglobin Disorders

to oxygen, and, even more importantly, the bin binding curve to the right. Except under
presence of one or two oxidized hemes per tet- clinical circumstances resulting in either acido-
ramer stabilizes the R quaternary structure and sis or alkalosis, plasma pH and red cell pH are
thereby increases the affinity of the remaining tightly regulated at 7.4 and 7.2, respectively. In
hemes for oxygen. Central nervous and cardio- contrast, levels of red cell 2,3-BPG can vary con-
vascular symptoms and signs begin to appear siderably in a wide range of clinical settings.
when methemoglobin exceeds 30%. Much low- Patients with low levels, such as those who
er levels are seen in individuals with congenit- have received large amounts of stored RBCs
al methemoglobinemia owing to deficiency in will have increased oxygen affinity, which could
cytochrome b5 reductase. Both toxic and con- compromise tissue oxygenation. Increased lev-
genital methemoglobinemia can be effectively els of RBC 2,3-BPG are much more commonly
treated by administration of the redox dye meth- observed, primarily in patients with some form
ylene blue. of hypoxia. As a result, the right shift in the
oxyhemoglobin dissociation curve significantly
enhances the unloading of oxygen to tissues and
is therefore a major mode of compensation,
Carbon monoxide (CO) binds to heme iron of particularly in patients with severe anemia.
hemoglobin with an affinity 210 greater than that
of oxygen. Normal red cells contain a very small
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