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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-
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ture and function of hemoglobin, along with the mechanism of assembly of its subunits,
as background for the rationale and basis of the different categories of disorders in the
classification.
n impressive degree of molecular engineer- tion to 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-
3
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 www.perspectivesinmedicine.org
Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011684
Cite this article as Cold Spring Harb Perspect Med 2013;3:a011684
1
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Press
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
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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-
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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
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
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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).
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-
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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
and concentration of HbS in the red cell. The does not participate at all in polymer forma-
kinetics of sickling are markedly concentration tion, this is sufficient to prevent sickling of all
dependent, the speed of the reaction being in- RBCs under physiologic conditions. This situa-
versely proportional to about the 30th power of tion is in contrast to what occurs when variable
the starting HbS concentration. Thus, a small amounts of HbF are present in HbSS disease or
increase in intracellular HbS concentration, the sickle/b-thalassemia syndromes. In the lat-
such as that resulting from loss of intracellular ter disorders, the HbF is distributed in a hetero-
water and cellular dehydration, can have a major cellular fashion, in which the HbF is present
effect on the speed of the polymerization reac- in only a subset of cells called F cells, leaving
tion. The amount and type of other non-S he- the other cells that lack HbF unprotected from
moglobins in the red cell can also influence the the antisickling effects of HbF. Nevertheless, a
extent or rate of sickling, and thus clinical se- marked increase in the proportion of F cells in
verity. HbSC disease is associated with signifi- such cases can have a beneficial effect on the
cant clinical manifestations. Why do SC pa- clinical manifestation of these sickling disor-
tients often have vaso-occlusive manifestations ders. Therefore, there is a great deal of interest
as dramatic as those with SS disease, whereas and ongoing research to develop strategies for
AS individuals have virtually no morbidity? enhancing HbF synthesis and F-cell production
HbC and HbA copolymerize to an identical de- as a therapeutic modality for sickling disorders
gree with HbS (Bunn et al. 1982). Two indepen- (see Sankaran and Orkin 2013).
dent factors conspire to make HbSC a disease.
The presence of HbC in the red cell greatly en-
UNSTABLE HEMOGLOBIN VARIANTS
hances potassium efflux and red cell dehydra-
tion, which increases corpuscular hemoglobin A substantial minority of Hb mutants have sub-
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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,
HEMOGLOBIN VARIANTS WITH
is clinically asymptomatic and is a notable ex-
ALTERED OXYGEN AFFINITY
ception to the general rule that clinical severity
is directly related to the amount (or %) of HbS Over 25 hemoglobin variants have been en-
in the RBC. In this condition, the HbF is dis- countered in individuals with erythrocytosis.
tributed in a pancellular fashion so that every Amino acid substitutions cause an increase in
RBC contains 30% HbF and, because HbF oxygen affinity usually because of impairment
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
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A 70 B
60
J-Balt
N-Balt
% of total hemoglobin
50 J-Iran
Korle-Bu
C
O-Arab
S
40 D-Los Ang
30 E
20 S-Oman
10
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
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proportion of seven positively charged b-subunit variants (blue) and three negatively charged variants (red).
(Updated from Bunn and McDonald 1983 and Bunn 1987.)
to oxygen, and, even more importantly, the bin binding curve to the right. Except under
presence of one or two oxidized hemes per tet- clinical circumstances resulting in either acido-
ramer stabilizes the R quaternary structure and sis or alkalosis, plasma pH and red cell pH are
thereby increases the affinity of the remaining tightly regulated at 7.4 and 7.2, respectively. In
hemes for oxygen. Central nervous and cardio- contrast, levels of red cell 2,3-BPG can vary con-
vascular symptoms and signs begin to appear siderably in a wide range of clinical settings.
when methemoglobin exceeds 30%. Much low- Patients with low levels, such as those who
er levels are seen in individuals with congenit- have received large amounts of stored RBCs
al methemoglobinemia owing to deficiency in will have increased oxygen affinity, which could
cytochrome b5 reductase. Both toxic and con- compromise tissue oxygenation. Increased lev-
genital methemoglobinemia can be effectively els of RBC 2,3-BPG are much more commonly
treated by administration of the redox dye meth- observed, primarily in patients with some form
ylene blue. of hypoxia. As a result, the right shift in the
oxyhemoglobin dissociation curve significantly
enhances the unloading of oxygen to tissues and
Carboxyhemoglobinemia
is therefore a major mode of compensation,
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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