Williams Thein 2018 Sickle Cell Anemia and Its Phenotypes

GG19CH06_Williams ARI 28 July 2018 12:37
Annual Review of Genomics and Human Genetics
Sickle Cell Anemia and Its

Phenotypes
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Annu. Rev. Genom. Hum. Genet. 2018.19:113-147. Downloaded from www.annualreviews.org
Thomas N. Williams1,2 and Swee Lay Thein3

1
Department of Epidemiology and Demography, KEMRI/Wellcome Trust Research
Programme, Kilifi, Kenya
2
Department of Medicine, Imperial College London, London W2 1NY, United Kingdom;
email: tom.williams@imperial.ac.uk
3
Sickle Cell Branch, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland 20892-1589, USA; email: sweelay.thein@nih.gov
Annu. Rev. Genom. Hum. Genet. 2018. 19:113–47 Keywords

First published as a Review in Advance on sickle cell anemia, genetics, genomics, genetic modifiers, Africa
April 11, 2018
The Annual Review of Genomics and Human Genetics Abstract

is online at genom.annualreviews.org
In the 100 years since sickle cell anemia (SCA) was first described in the med-
https://doi.org/10.1146/annurev-genom-083117- ical literature, studies of its molecular and pathophysiological basis have been
021320
at the vanguard of scientific discovery. By contrast, the translation of such
Copyright c 2018 by Annual Reviews. knowledge into treatments that improve the lives of those affected has been
All rights reserved
much too slow. Recent years, however, have seen major advances on sev-
eral fronts. A more detailed understanding of the switch from fetal to adult
hemoglobin and the identification of regulators such as BCL11A provide
hope that these findings will be translated into genomic-based approaches
to the therapeutic reactivation of hemoglobin F production in patients with
SCA. Meanwhile, an unprecedented number of new drugs aimed at both the
treatment and prevention of end-organ damage are now in the pipeline, out-
comes from potentially curative treatments such as allogeneic hematopoietic
stem cell transplantation are improving, and great strides are being made in
gene therapy, where methods employing both antisickling β-globin lentivi-
ral vectors and gene editing are now entering clinical trials. Encouragingly,
after a century of neglect, the profile of the vast majority of those with SCA
in Africa and India is also finally improving.
113
INTRODUCTION
Sickle cell anemia (SCA) has been recognized in Africa for generations; however, it was not formally
described in the western literature until November 1910, when Herrick (93) reported a case of
anemia that was associated with “peculiar elongated and sickle-shaped red blood corpuscles”
on microscopy in a dental student from Grenada. SCA was subsequently heralded as the first
“molecular disease” when Pauling et al. (149) ascribed its basis to the presence of an abnormal
hemoglobin, hemoglobin S (HbS), in 1949. In 1957, Ingram (100) discovered that HbS was
caused by a single amino acid substitution (glutamic acid changed to valine) at position 6 of the
β-globin chain of hemoglobin, and in 1963, Goldstein et al. (83) showed that this amino acid
substitution arose from a single base change (A>T) at codon 6 (rs334). SCA, therefore, has always
been at the forefront of molecular medicine and arguably launched the whole field of human
molecular genetics. Because of this disease’s genetic simplicity, being caused by a single change
in a single gene, it has been used to illustrate and validate many of the advances in this field,
including proof-of-principle studies in DNA diagnostics, predictive genetics, and population and
epidemiological genetics (104, 146, 167, 171). The contribution of genetic modifiers to its extreme
clinical heterogeneity made SCA an exemplar of the effects of genetic background on a single-gene
disease. More recently, SCA has demonstrated the potential for genome-wide association studies
in the discovery of interacting genes that might be of clinical or therapeutic significance. Genome-
wide association studies of HbF, a quantitative trait that influences SCA severity, identified two
quantitative trait loci that are not linked to the HBB cluster: BCL11A and HBS1-MYB (122, 195).
These results have led to various genetic approaches to the reactivation of HbF that are now being
explored as therapeutic options in SCA (96). Figure 1 shows a time line of key genetic discoveries
and significant events in SCA.
Given its important role in scientific discovery, the process of translating such knowledge into
new treatments has been far too slow, and for more than 30 years only two approaches to disease
modification—chronic blood transfusion and hydroxyurea therapy—have been widely deployed.
However, more recently, a sea change in the therapeutic landscape has led to the development of
numerous new drugs that target different aspects of the pathophysiology of SCA (189).
THE MOLECULAR BASIS OF SICKLE CELL DISEASE

The term sickle cell disease (SCD) describes a clinical syndrome caused by the presence of HbS.
The genetic causes of SCD include homozygosity for the rs334 mutation (HbSS) (generally
known as SCA) and compound heterozygosity between rs334 and mutations that lead to either
other structural variants of β-globin (such as HbC) or reduced levels of β-globin production (β-
thalassemia). In patients of African ancestry, SCA is the most common cause of SCD (65–70%),
followed by HbSC (about 30%), with HbS/β-thalassemia being responsible for the majority of
the balance. Although all forms of SCD are important, SCA is by far the best described and is the
sole focus of this review.
THE ORIGINS, GLOBAL BURDEN, AND DISTRIBUTION OF SICKLE

CELL ANEMIA
The heterozygous status of rs334 (coding for HbS) results in the sickle cell trait (HbAS), in which
erythrocytes contain a mixture of both normal HbA and HbS molecules. Through mechanisms
that have not been completely elucidated, such heterozygotes have a substantial survival advan-
tage in malaria-endemic environments. Large-scale genetic studies have recently revealed the
extraordinary degree of this protection. In a study of almost 12,000 severe malaria cases and more
114 Williams · Thein

1963 1978 1995 2010 2016

Discovery that Prenatal diagnosis of Publication of MSH Successful addition • SUSTAIN study of
Gluβ6Val arises from SCA using restriction study of hydroxyurea of HbAT87Q in gene crizanlizumab; DOVE and
an A>T substitution fragment length (Charache et al. 1995) therapy for βE/β0- MAGiC studies of prasugrel
at codon 6 (Goldstein polymorphism (Kan thalassemia and magnesium; phase 2
et al. 1963) & Dozy 1978) 1998 2007 study of GBT440
FDA approval of Use of induced
hydroxyurea in adults pluripotent stem • Initiation of treatment of
cells for gene seven SCD patients with
1910 1957 1984 1999 therapy in sickle HbAT87Q
First reported Discovery that First allogeneic STOP study of mice; GWAS
case of SCA Gluβ6Val causes HSCT in SCD stroke prevention identification of • TWiTCH study of
(Herrick 1910) HbS (Ingram 1957) BCL11A HbF locus hydroxyurea
00
30
50
70
90
00
10
20
19
19
19
19
19
20
20
20
1846 1949 1985 2000 2015 2018

First apparent Publication of PCR for direct Beginning of genetic First successful Discovery that
case of SCD studies of SCA as a detection of association studies treatment of the βS-globin
molecular disease SCA for SCA SCD patient mutation had a
(Pauling et al. 1949), 1996 2006 with HbAT87Q single origin
SCA as a recessive Establishment of Development of
disease (Neel 1949), allogeneic HSCT gene therapy
and the genetics of in SCD vector
SCA (Beet 1949)
1982 1990–2000s 2011 2017
Direct detection of the Development of STOP II study Use of allogeneic
βS-globin mutation by transgenic sickle for stroke HSCT in 1,000
restriction enzyme mouse models prevention SCD patients
analysis (Wilson et al.
1982)
Figure 1
Time line of key discoveries and significant events in SCA. Abbreviations: DOVE, Determining Effects of Platelet Inhibition on
Vaso-Occlusive Events; FDA, US Food and Drug Administration; Gluβ6Val, substitution of valine for glutamic acid at position 6 of
the β-globin chain of hemoglobin; GWAS, genome-wide association study; HbA/F/S, hemoglobin A/F/S; HSCT, hematopoietic stem
cell transplantation; MAGiC, Magnesium for Children in Crisis; MSH, Multicenter Study of Hydroxyurea in Sickle Cell Anemia; SCA,
sickle cell anemia; SCD, sickle cell disease; STOP, Stroke Prevention Trial in Sickle Cell Anemia; SUSTAIN, Study to Assess Safety
and Impact of SelG1 with or without Hydroxyurea Therapy in Sickle Cell Disease Patients with Pain Crises; TWiTCH, Transcranial
Doppler with Transfusions Changing to Hydroxyurea.
than 17,000 controls conducted throughout Africa, Asia, and Oceania, HbAS conferred an 86%
reduction in the risk of severe malaria (odds ratio 0.14, 95% confidence interval 0.12–0.16) at a
significance level (P = 1.6 × 10−225 ) rarely seen in epidemiological studies (118). Furthermore,
by preventing malaria, HbAS protects from consequences of malaria such as undernutrition and
fatal bacterial infections (172). These wide-ranging benefits have driven rs334 to high population
frequencies throughout much of the historically malaria-endemic world, so that carrier frequen-
cies of 10–20% are now commonplace in many parts of sub-Saharan Africa, India, the Middle
East, and the Mediterranean basin (152) (Figure 2). Genetic analyses had suggested that rs334
arose and was amplified by selection separately in multiple populations (73), but a recent paper by
Shriner & Rotimi (176a) has now provided evidence that it had a single origin 7,300 years ago in
West Africa (see the Note Added in Proof at the end of this article).
These high population frequencies mean that a significant proportion of babies born within
these geographic areas are affected by SCA. Furthermore, recent global population migration
means that SCD is now a major contributor to health problems in many regions that are well be-
yond its early geographic origins (154), including the Caribbean islands, Brazil, the United States,
the United Kingdom, France, and cosmopolitan parts of other European countries (154). Recent
estimates suggest that globally, more than 300,000 children are born with SCA every year (88, 153).
In addition to HbS, malaria has led to positive selection for a wide range of other genetic con-
ditions in the same geographic areas. Like HbS, the best described of these conditions also relate
www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 115

HbS allele frequency (%)

0–0.51
0.52–2.02
2.03–4.04
4.05–6.06
6.07–8.08
8.09–9.60
9.61–11.11
11.12–12.63
12.64–14.65
14.66–18.18
Malaria endemicity
Malaria free
Epidemic
Hypoendemic
Mesoendemic
Hyperendemic
Holoendemic
Figure 2
The global distribution of sickle cell anemia in relation to the historical distribution of Plasmodium falciparum malaria. (a) The
geospatial distribution of the HbS allele; these data were derived by using a Bayesian geostatistical model with data from published and
unpublished population surveys for allele frequencies. (b) The historical distribution of malaria based on Reference 115. Abbreviation:
HbS, hemoglobin S. Figure adapted from Reference 152.
to erythrocytes, including other structural hemoglobin variants, the thalassemias, erythrocyte en-
zymopathies (most notably glucose-6-phosphate dehydrogenase deficiency), and polymorphisms
in erythrocyte surface molecules, including the Duffy antigen receptor for chemokines (DARC),
complement receptors, and ABO blood groups (216). As a result, many patients with SCA also
inherit other genetic variants, some of which may affect their clinical phenotype, the relevance of
which we discuss in further detail below (see the section titled Factors Determining Outcome).
DIAGNOSIS
Preconception, antenatal, and neonatal screening programs are important in the clinical care
and public health management of SCA. The most common approaches to diagnosis in
high-income countries include gel-based or capillary electrophoresis, high-performance liquid

chromatography, and isoelectric focusing. All are sensitive in identifying affected individuals but
are limited by their inability to reliably distinguish HbSS from HbS/β0 -thalassemia. However,
most neonatal screening programs involve testing during the first week of life, when the expres-
sion of adult hemoglobin (and, therefore, HbS) is still low, which leads to problems with reduced
sensitivity. This has prompted the development of new approaches that include tandem mass spec-
trometry (126), DNA diagnostics (including Taqman PCR and sequence analysis of specifically
amplified HBB genes) (192), and next-generation sequencing analysis (163, 176). Although these
increasingly sophisticated methods are now entering clinical practice in high-income countries,
few are readily available to the majority of patients in low-income regions. For many years, the
mainstay of diagnosis in these regions has been the sodium metabisulfite sickling test or commercial
variations of it (94); however, these tests cannot reliably distinguish carriers from affected patients.
Several alternative, point-of-care approaches are currently under development, of which the most
advanced—an antibody-based lateral-flow assay marketed under the name of Sickle SCAN—is
now commercially available (105).
PATHOPHYSIOLOGY
The fundamental event that underlies the manifold pathophysiological consequences of SCA is the
polymerization of HbS under conditions of low oxygen tension (35). HbS polymerization alters
both the structure and function of erythrocytes, initiating a cascade of events that ultimately affect a
wide range of tissues, with far-reaching clinical and pathological consequences. Our understanding
of these processes has been accelerated more recently through newer techniques, of which the use
of murine models has been particularly influential (225). The most important of these processes
are summarized in Figure 3 and described in further detail below.
Vasoocclusion
The polymerization of HbS activates calcium-influx-mediated dehydration, increasing the concen-
tration of intracellular hemoglobin, further exacerbating polymerization, and ultimately resulting
in irreversibly distorted and bizarrely shaped erythrocytes (181). Sickled erythrocytes have diffi-
culty negotiating the microvasculature and cause logjams that result in local tissue hypoxemia. The
recent development of microfluidic devices provides a graphic in vitro illustration of how this sim-
ple mechanical process might lead to tissue ischemia in vivo (62). Nevertheless, it would be overly
simplistic to attribute the tissue injury that accompanies SCA to mechanical obstruction alone.
The sickled erythrocytes are also highly fragile (life span of 16 days compared with 120 for normal
red blood cells) (90), and the continual release of cell-free hemoglobin from hemolysis depletes
the bioavailability of nitric oxide (NO), contributing to pathology. Neutrophilia is an additional
feature of SCA that has been consistently correlated with disease severity (38, 107, 141, 156, 225),
and more recent studies have shown that even at steady state, these neutrophils are highly activated
(109, 225). The detrimental effect of neutrophils in sickle pathophysiology is supported by case
reports of severe and fatal acute events triggered by drug-induced neutrophil production (1, 6, 86,
208). Recent studies indicate that neutrophils play a central role in vasoocclusion through their
interactions with both erythrocytes and endothelium. Important elements include the activation
of endothelial cells to upregulate the expression of cytoadhesion molecules such as the P- and
E-selectins, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1
(VCAM-1). Endogenous factors of tissue activation include the release of proinflammatory cy-
tokines by monocytes (including TNF-α, IL-6, IL1-β, and IL-3), while exogenous factors include
microbial antigens (95). Observations made in mice have shown that sickled erythrocytes bind to

HbS Polymerized HbS
Oxygenated RBC Deoxygenated RBC Dehydrated sRBC
Microbial
antigen NET
Neutrophil
Vasoocclusion
RBC Monocyte
Cytokines
Inflammation
Hemolytic Neutrophil adhesion

molecule Hemolysis Reperfusion
RBC
Heme Neutrophil NO binding

Heme
ROS
sRBC Heme Endothelial
permeability
Platelet
Endothelial
TLR4 adhesion molecules
Endothelial cell VWF Selectin
Figure 3
Pathophysiology, inflammatory stimuli, and cellular interactions in SCA. Polymerization of deoxy-HbS eventually leads to the
formation of dehydrated and misshapen erythrocytes, typically with a sickled shape. The sickled cells trigger microvascular occlusion
through interactions with activated neutrophils and platelets and adhesion to vascular endothelium, leading to ischemia and tissue
hypoxia followed by vasodilation and reperfusion injury. The damaged erythrocytes are short lived, continuously releasing hemoglobin,
and oxidized hemoglobin releases heme. Heme functions as a damage-associated molecular pattern that activates endothelial cells,
macrophages, and neutrophils and promotes the formation of NETs (NETosis) via TLR4 binding. Abbreviations: HbS, hemoglobin S;
NET, neutrophil extracellular trap; NO, nitric oxide; RBC, red blood cell; ROS, reactive oxygen species; SCA, sickle cell anemia;
sRBC, sickle red blood cell; VWF, von Willebrand factor.
neutrophils that are themselves bound to inflamed cremasteric venules, resulting in vasoocclusion
(194). Moreover, the finding that mice deficient in P- and E-selectin are protected from vasooc-
clusion has provided impetus for the development of treatments designed to block this interaction
(194). In support of this idea, neutrophils from HbSS individuals show greater levels of obstruction
compared with those from HbAA individuals in E-selectin/ICAM-1-coated microchannels (58).
HbSS erythrocytes alone were nonadherent but required mixed suspensions of erythrocytes and
neutrophils to elicit vasoocclusion (58). A recent study showed that neutrophils from SCA individu-
als can also form neutrophil extracellular traps that inflict further damage on the endothelium (45).
In addition to neutrophils and monocytes, platelets are activated in steady-state patients with
SCA, and activation increases further during vasoocclusive crises (101, 206, 225). Platelets form

aggregates with erythrocytes, monocytes, and neutrophils both in patients and in murine models
(57, 75, 221, 225). Moreover, as with neutrophils, it appears that this process is dependent on
P-selectin (159, 161). SCA has long been considered a hypercoagulable state, as illustrated by
the increased rates of venous and arterial thrombotic events. Not only is the incidence of venous
thrombotic events increased (particularly pulmonary embolism), but these events also occur in
younger patients and are significantly associated with elevated steady-state platelet counts (34, 198).
This has prompted clinical trials on antiplatelet agents for the prevention of acute vasoocclusive
pain (below), and the hospital management of acute pain crises in many centers now includes
prophylactic anticoagulation. Because patients who have suffered one venous thrombotic event
are more likely to have additional events, it has been proposed that such patients should be placed
on lifelong anticoagulation.
Hemolysis
Patients with SCA are in a constant state of uncompensated hemolysis, which further increases
during painful crises, as reflected by further elevated levels of plasma lactate dehydrogenase (16,
77). While steady-state hemolysis involves both an intravascular and an extravascular component
(23), the intravascular element has been the main focus from a pathophysiological perspective. It
has been proposed that the release of both free hemoglobin and arginase is directly responsible for
a chain of events that results in vascular pathology. Important elements include the depletion of
bioavailable NO via a dioxygenation reaction (165), the depletion of arginine (a substrate for NO
production), and the promotion of oxidant stress and endothelial inflammation through exposure
to free heme and heme-laden microparticles (36). Exposure of endothelium to heme in mice leads
to upregulation in the production of various adhesion molecules, including ICAM-1, P-selectin,
and fibronectin; the recruitment of neutrophils; and increased vascular permeability (207). Such
observations have led to the hypothesis that excessive heme might play a role in exacerbating the
vasoocclusive process. Free heme also leads to the activation of neutrophils and the formation of
neutrophil extracellular traps, which damage the vasculature (45). As a result of such observations,
it has been postulated that variation in the degree of hemolysis among individual patients may
contribute to the variable phenotypes of SCA (106).
CLINICAL COURSE AND COMPLICATIONS

The clinical course of SCA is notoriously variable. Progressive damage to a range of organs is a
constant feature of the disease; however, the factors that determine the degree to which specific
organs are involved remain incompletely understood. Historically, the most widely recognized
complications of SCA were the acute clinical events that punctuate periods of reasonable health,
of which pain is the most common. However, with increasing life expectancy, many complications
that result from damage to a range of end organs are now being described. The most common com-
plications of SCA are summarized in Table 1 and Figure 4 and discussed in further detail below.
Acute Manifestations of Sickle Cell Anemia

One of the most common early manifestations of SCA is the hand–foot syndrome, which is
characterized by the painful swelling of one or more extremities owing to the infarction of marrow
within the small bones of the hands and feet (223). Beyond the first year of life, the most frequent
acute complication is a painful crisis. Such episodes most commonly occur in the extremities, chest,
abdomen, and back and result from vasoocclusive episodes with ischemia–reperfusion injury (67).

GG19CH06_Williams
Table 1 Commonly recognized complications of sickle cell anemia

Complication Definition Age Presumed etiology Incidence/prevalence Reference(s)
ARI
Acute
120
Hand–foot Painful swelling of one or more 0–3 years Bone marrow infarction secondary 31.3/100 PYO in 80, 223
syndrome extremities to vasoocclusion children age
6–12 months
Williams
Painful crisis Acute pain, most commonly in >6 months but Vasoocclusion, hypoxia, and ∼40 episodes/100 PYO 67, 80
28 July 2018
·
the extremities, chest, occurs throughout ischemia–reperfusion injury beyond the second year
abdomen, or back life of life
Thein
12:37
Acute infections Severe bacterial infections, Childhood Immune compromise, including 10/100 PYO during 80
including septicemia, functional asplenia, vasoocclusion early childhood
meningitis osteomyelitis, and in barrier tissues, and bone (without prophylaxis)
septic arthritis infarction
Acute chest Acute onset of respiratory Increasing frequency Vasoocclusion, bone marrow fat 24.5/100 PYO in young 38, 203
syndrome symptoms with features from early embolism, and microembolism of children; 8.8/100 PYO
similar to pneumonia childhood; occurs aggregated blood cells in older adults
throughout life
Acute anemia A decline in hemoglobin of Most common in Sequestration (most commonly in ∼10/100 PYO in 80
2 g/dL or more from childhood spleen or liver), transient red cell children
steady-state values aplasia secondary to parvovirus
B19 infection, and posttransfusion
hemolysis
Priapism Unwanted painful and Increasing frequency Sequestration 20–89% lifetime 5, 114, 143
sustained erection of the penis from early prevalence in boys and
for more than 4 hours, often childhood men
recurrent or persistent
Stroke Acute cerebrovascular accident Increasing frequency Infarctive stroke secondary to 0.61/100 PYO during 141
with age vasculopathy of cerebral vessels childhood (without
(childhood) or hemorrhagic stroke specific intervention)
(more common in adulthood)
Acute renal injury Acute deterioration in renal Any age Sequestration 8% prevalence in 110
function children with acute
chest syndrome
(Continued)
GG19CH06_Williams
Table 1 (Continued)
Complication Definition Age Presumed etiology Incidence/prevalence Reference(s)
ARI
Chronic
Silent cerebral Clinically silent lesions of 3 mm Increasing frequency Vasoocclusion and tissue infarction 39% during childhood 24
infarcts or more on magnetic with age
resonance imaging scanning
28 July 2018
Pulmonary Mean pulmonary artery Increasing frequency 50% precapillary (vasculopathy of 6.0–10.4% prevalence 74, 121, 147
hypertension pressure of ≥25 mm Hg at with age pulmonary arteries, sequestration, during adulthood
rest, measured by right heart and microemboli), 50%
12:37
catheterization postcapillary secondary to left

ventricular dysfunction
Avascular necrosis Avascular necrosis of any bone, Increasing frequency Vasoocclusion >20% lifetime 160
of bones most commonly the femoral with age prevalence
head and shoulder girdle
Heart failure Most commonly left ventricular Increasing frequency Ventricular hypertrophy and high Universal to some 131
failure with age output failure secondary to degree in adults over
anemia age 30
Renal failure Deteriorating renal function, Increasing frequency Vasoocclusion Advanced disease (stage 173
reduced concentrating ability, with age III–IV) in 4–18% of
proteinuria, and progressive adults
renal failure
Leg ulcers Chronic skin ulcers, most Increasing frequency Hemolysis with vasculopathy >14% lifetime 160
commonly around the with age prevalence
malleolar regions
Cholelithiasis Gallstones and gallbladder Increasing frequency Chronic hemolysis >28% lifetime 160
disease with age prevalence
Hypersplenism Enlargement of the spleen with More common in Unknown, but commonly follows 20% cumulative 160
persistent reduction in childhood an episode of splenic sequestration prevalence
steady-state hemoglobin and
www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes

multiple blood cell types
121
Abbreviation: PYO, person-years of observation.
Hemorrhagic and Cardiac: pulmonary

infarctive strokes hypertension
Sickle hepatopathy Sickle nephropathy
Priapism
Avascular necrosis
Bone infarction Leg ulceration

osteomyelitis
Figure 4
Common complications of SCA that add to morbidities. While many complications are related to age, it is also clear that some patients
are genetically predisposed to some complications. Predisposing or protective genetic variants have been identified for some
complications, such as bilirubin levels and gallstones, as well as sickle nephropathy. Clearly some patients are also predisposed to stroke,
avascular necrosis, and pulmonary complications, but genetic variants for these complications need genetic and functional validation.
Abbreviation: SCA, sickle cell anemia.

At times, painful crises can be complicated by bony necrosis with secondary infections, including
osteomyelitis and septic arthritis. Rare in the first six months of life (because fetal hemoglobin
levels are still elevated), painful crises become increasingly frequent thereafter, reaching an overall
incidence of approximately 40 per 100 person-years of observation (PYO) beyond the second
year of life (80). Painful crises frequently require treatment with strong analgesics, including
opioids, with the attendant risk of the development of opioid dependency. Another common acute
complication of SCA is acute chest syndrome (ACS). ACS presents with a clinical picture (including
cough, shortness of breath, and signs of consolidation and hypoxemia) that is difficult to distinguish
from acute pneumonia (223). Episodes can present in apparent isolation or complicate painful crises
or surgical procedures, particularly those involving the abdomen (202, 203). Common etiological
factors include acute infections, bone marrow fat embolism, and microembolism of aggregated
blood cells (132, 223). The incidence of ACS is lower in older adults than in children (38, 203),
but both severity and mortality are higher in adults, largely because of a higher incidence of bone
marrow and fat emboli in adults (202) and the frequent presence of comorbidities.
Acute anemia, usually defined as a decline in hemoglobin of 2 g/dL or more from steady-state
levels (223), is a common feature of painful crises but can also result from sequestration of ery-
throcytes in deep vascular beds, most commonly within the spleen (acute splenic sequestration)
or liver (acute hepatic sequestration) (27, 89); aplastic episodes secondary to parvovirus B19 in-
fection (174); or increased hemolysis secondary to serious acute infections or delayed transfusion
reactions. Patients with SCA are also at substantial risk of serious acute bacterial infections, partic-
ularly those caused by Streptococcus pneumoniae, Haemophilus influenzae, and non-Typhi Salmonella
species. Progressive involution of the spleen leads to functional asplenia from early childhood,
vasoocclusive episodes can violate the integrity of gut and other barrier tissues to result in the
translocation of pathogens, and ischemic tissue in various organ systems can provide a niche for
septic foci (31). In the preprophylaxis era, the incidence of invasive bacterial sepsis was as high as
10 per 100 PYO in the first 5 years of life (80).
Three further common acute complications are priapism, acute kidney injury, and stroke.
Priapism, an unwanted and painful erection of the penis that lasts for more than 2 h, is frequently
recurrent and occurs with a lifetime prevalence of 20–89% in boys and men (5, 114, 143). Acute
kidney injury is an underrecognized acute event that in one prospective case series complicated
8% of all admissions with ACS in children (110). Finally, stroke is probably the most serious
and devastating acute complication of SCA, occurring at an overall rate of 0.61 episodes per 100
PYO before the development of specific interventions (141). Strokes can be either infarctive or
hemorrhagic, the former being the more common (141). During childhood, the risk of infarctive
stroke can be predicted from blood velocities in the cerebral vessels measured by transcranial
Doppler (TCD) ultrasonography (2). As a result, TCD is now widely deployed for screening, and
those with increased velocities (≥200 cm/s) are deemed at high risk of developing a stroke and
targeted for preventive transfusion therapy (see the section titled The Management and Outcome
of Sickle Cell Anemia in High-Income Countries).
Chronic Complications of Sickle Cell Anemia

With increasing survival, cumulative complications of SCA are becoming increasingly apparent
in adults in high-income countries. It is now clear that subtle neurological deterioration is more
common than previously realized (54). Neuroimaging studies, most recently using near-magnetic
resonance imaging, have shown that silent cerebral infarcts (signal abnormalities of at least 3 mm in
the presence of a normal neurological examination) are common, affecting 39% of patients by the
age of 18 years in one such study (24). Risk factors include lower hemoglobin concentrations and

higher systolic blood pressures, male sex, and acute anemic events (25, 55). Chronic heart and lung
complications are also common, particularly in older children and adults. Clinical manifestations
include impaired pulmonary function and exercise tolerance and progressive heart failure (85).
Pulmonary hypertension, defined as a mean pulmonary artery pressure of ≥25 mm Hg at rest as
determined by right heart catheterization, is the most common chronic pulmonary complication of
SCA and a major cause of morbidity and mortality in older adults. Three studies utilizing right heart
catheterization estimated the prevalence of pulmonary hypertension in adults at 6.0–10.4% (74,
121, 147). In studies conducted in France and Brazil, pulmonary hypertension was associated with
three-year mortality rates of 12.5% and 37.5%, respectively (74, 147), while a six-year mortality
of 37% was reported in a study conducted in the United States (121). Tricuspid regurgitant jet
velocity has been used as a noninvasive alternative to right heart catheterization in the prediction of
pulmonary hypertension in multiple studies. Tricuspid regurgitant jet velocity values of ≥2.5 m/s,
as measured by two-dimensional echocardiography, are suggestive of pulmonary hypertension, and

values of ≥3 m/s are strongly predictive (74, 147). Approximately half of pulmonary hypertension
cases are precapillary and result from a vasculopathy caused by recurrent sickling, inflammation,
chronic thromboembolism, and hemolysis-induced NO depletion, as discussed above (85). The
rest have postcapillary disease from left ventricular dysfunction caused by elevated cardiac output
and left ventricular volume overload secondary to chronic anemia (131).
The hypoxia, acidosis, and hyperosmolarity within the inner medulla of the renal cortex create
a perfect environment for the polymerization of HbS. Over time, repeated cycles of acute kid-
ney injury lead to the chronic microvascular disease of established sickle cell nephropathy. Renal
complications are manifest from early life (213) and include hyperfiltration, hyposthenuria (dimin-
ished concentrating ability), and albuminuria. In childhood, sickle cell nephropathy can manifest
as polyuria, nocturia, and nocturnal enuresis. The prevalence of albuminuria increases with age,
and in one study 60% of patients with SCA had microalbuminuria by age 55 (50). Advanced
chronic kidney disease (stage III–IV, defined by an estimated glomerular filtration rate of 15–
60 mL/min per 1.73 m2 ) occurs in 4–18% of adults with SCA (173). Increasing renal dysfunction
can also result in arterial hypertension. Table 1 summarizes additional common acute and chronic
complications of SCA.
FACTORS DETERMINING OUTCOME

Both environmental and genetic factors contribute to the clinical variability of SCA. Although the
importance of weather as a trigger of acute pain has been suspected for many years, it has been
difficult to prove for logistic reasons (155). Twin studies suggest that environmental factors in
general are important determinants of the clinical course of SCA. Such studies include two case
reports limited to single pairs, one with HbSS and α-thalassemia and the other with HbS and β-
thalassemia (8, 103), and nine pairs of identical twins from Jamaica, six with HbSS and three with
HbSC (214). These studies show that, while twins may have similar laboratory parameters and
attain similar heights and weights, there is considerable discordance in the frequency of painful
crises and other complications. Furthermore, other environmental factors, including nutritional
status and access to social support and medical care, influence risk factors such as infections (155).
Factors that influence the primary event of HbS polymerization (including coexisting α-
thalassemia and HbF concentrations) have a global effect on the disease phenotype. Approxi-
mately one-third of SCA patients of African descent also have α-thalassemia, usually caused by
the common African 3.7-kb (−α3.7 ) deletional variant (200). While the majority are heterozygous
(αα/−α3.7 ), 3–5% are homozygous (−α3.7 /−α3.7 ). Coexisting α-thalassemia results in reduced
intracellular HbS and mean corpuscular hemoglobin concentrations, reductions in the frequency

of HbS polymerization and number of irreversibly sickled cells, and an increased hematocrit (68,
180). While some of these effects are beneficial, others are detrimental. Patients with SCA and
α-thalassemia have a reduced risk of hemolytic complications, such as pulmonary hypertension,
cardiomyopathy, nephropathy, priapism, and leg ulcers, but an increased risk of vasoocclusive
complications, such as ACS, painful crises, osteonecrosis, and retinopathy (179). Several stud-
ies have also demonstrated an association with lower TCD velocities and stroke (26, 49, 71).
The reduced hemolysis reduces bilirubin, with a quantitative effect that is independent of that
of the UGT1A1 promoter polymorphism (200). However, coinheritance of α-thalassemia blunts
the response to hydroxyurea therapy, potentially because it affects the HbF and mean corpuscular
volume responses (199).
HbF has a sparing effect on HbS polymerization because the hybrid tetramers (α2 γβS ) and
HbF (α2 γ2 ) have a diluting effect on the intracellular HbS (α2 βS 2 ) concentration. In addition,
α2 γβS tetramers cannot partake in HbS polymerization. Even a small decrease in HbS concen-
tration can increase the delay time to polymerization and allow more cells to escape sickling (129,
158). As a result, HbF has a globally beneficial effect on SCA: High levels are associated with
reduced pain and improved survival (156), while low levels are associated with an increased risk of
brain infarcts in young children (209). Genome-wide association studies of HbF and its associated
trait, erythrocytes containing HbF (or F cells), have shown that genetic variants at three quan-
titative trait loci—BCL11A on chromosome 2p, HMIP-2 on chromosome 6q, and Xmn1-HBG2
(rs7482144) on chromosome 11p—together account for up to half of the common variation in
the Caucasian non-SCA population (191) and between 8% and ∼20% of the variability in HbF
concentrations among patients with SCA (112, 113, 117, 219). Downstream studies in primary
human erythroid progenitor cells and transgenic mice have demonstrated that BCL11A acts as
a repressor of γ-globin gene expression (169, 222). High-resolution mapping, further validated
by systematic dissection using the CRISPR/Cas9 genome-editing approach, has confirmed that
these variants are localized to enhancers in intron 2 of the gene that are erythroid specific (18).
With regard to HMIP-2 on chromosome 6q, subsequent studies showed that the intergenic
region contains two clusters of HbF-affecting variants that occupy critical core enhancer elements
that regulate MYB, which encodes the MYB transcription factor essential for hematopoiesis and
erythroid differentiation (177). As a result, it has been proposed that MYB modulates HbF expres-
sion by two potential mechanisms: (a) indirectly, by altering the kinetics of erythroid differentia-
tion (low MYB levels accelerate erythroid differentiation and lead to the release of early erythroid
progenitor cells that are still synthesizing predominantly HbF) (102, 178), and (b) directly, via ac-
tivation of KLF1 and other repressors of γ-globin genes (29, 184). Rare variants in KLF1 have also
been associated with HbF levels in various erythrocyte disorders, including SCA (151). However,
several genome-wide association studies of HbF have failed to identify common KLF1 variants (28,
130). In African American patients and patients from Cameroon, an association between HbF and
three loci (BCL11A, HBS1L-MYB, and XmnI-HBG2) also correlated with reductions in the rates
of acute vasoocclusive pain and hospitalization (113, 219), while the presence of HbF-promoting
variants in BCL11A was associated with a reduced rate of SCA complications, particularly with
stroke in a Brazilian cohort (112).
Coexisting α-thalassemia and HbF concentrations alone are not sufficient to explain the ob-
served diversity in the severity of SCA between individual patients. Both candidate gene and
genome-wide association studies have been used to search for other modifying genetic variants.
Many of these studies have focused on specific complications of SCA (such as stroke, proteinuria,
osteonecrosis, or pulmonary hypertension) as separate phenotypic end points; however, given the
complexity of the factors that lead to such outcomes, it is not surprising that studies that have
employed more simple intermediate phenotypes that are reproducible, measurable, and disease

related (like HbF and bilirubin levels) have been more successful. Based on an understanding of
SCA pathophysiology, candidate genes that could plausibly influence different SCA-related com-
plications have been selected and single-nucleotide polymorphisms (SNPs) identified through
association studies. The complications studied included acute painful episodes, bilirubin levels,
and predisposition to gallstones, stroke, priapism, osteonecrosis (or avascular necrosis), leg ulcers,
pulmonary disease (pulmonary hypertension and ACS), bacteremia and other infections, and re-
nal disease. Table 2 summarizes the genetic variants that have been associated with these major
SCA-related complications.
Rates of acute pain can vary widely among patients, but the highest rates are in patients with
high hematocrits and low HbF concentrations (17). Besides HbF levels and a possible role of α-
thalassemia, very little is known about the genetic basis for the variability of painful crises. Case–
control studies are problematic because it is difficult to objectively quantitate both the frequency
and severity of painful episodes within or among patients. The response to opioid analgesia varies
considerably among individuals, and the efficacy of these drugs may itself be related to genetic
variability in their metabolism (17). An association between serum bilirubin levels or gallstones and
a promoter polymorphism in UGT1A1 has been consistently demonstrated in SCA individuals of
all ages and different population groups using both candidate gene and genome-wide approaches
(42, 70, 124, 200). The influence of polymorphisms in UGT1A1 became more evident in patients
while on hydroxyurea therapy: Children with the 6/6 UGT1A1 genotype achieved normal bilirubin
levels, while children with 6/7 or 7/7 UGT1A1 genotypes did not (92). Co-occurrence of α-
thalassemia also has a quantitative effect on serum bilirubin levels in patients with SCA (200). The
triad of the UGT1A1 genotype, SCA, and gallstones presents a possible clinical context where
genetic information might aid clinical decision-making with regard to elective cholecystectomy.
A familial predisposition to stroke was first suggested by an increased incidence among siblings
with SCA (61), prompting numerous genetic studies. In some, stroke was subdivided into large-
and small-vessel disease based on imaging studies (97). Of the 38 published SNPs that have been
associated with stroke, the effects of α-thalassemia and SNPs in four genes (ADYC9, ANXA2, TEK,
and TGFBR3) have been most replicable, albeit at marginal levels of significance (71). Genome-
wide association studies and whole-exome sequencing studies have recently identified additional
protective associations in pediatric patients at two genes (GOLGB1 and ENPP1), but, again, this
warrants further validation (72).
Independent studies have identified associations between mutations in KLOTHO (KL) and
priapism in different populations (66, 139). Separately, the TGF-β/SMAD pathway has also been
implicated in priapism risk (66). Higher hematocrits are a predisposing factor for osteonecrosis,
and hence an increased incidence has been seen in patients with coexisting α-thalassemia. Studies
suggesting that factors in the coagulation pathway, such as MTHFR and platelet adhesion (HPA-5B
allele), may also be involved have been inconclusive (9, 15, 39, 108, 196).
Leg ulceration varies widely in SCA patients, with major apparent differences in geo-
graphical prevalence. It affects up to 70% of Jamaican patients, but reported rates have been
much lower (30–40%) in other patient cohorts (7). This complication is closely associated
with the severity of hemolysis, which potentially explains the protective effect of co-occurring
α-thalassemia. Candidate gene association studies have implicated several genes in the TGF-
β/SMAD/BMP pathway (138). Polymorphisms in DARC have also been associated with the per-
sistence of leg ulcers, and it has been suggested that the relatively higher white cell and neutrophil
counts potentiate inflammation in the Duffy-positive patients (60).
Pulmonary hypertension, defined by a tricuspid regurgitant jet velocity of ≥2.5 m/s, occurs in
approximately 30% of adults with SCA and is an important risk factor for premature death (81).
Multiple gene associations have been identified, including polymorphisms in the TGF-β/BMP

Table 2 Published genetic associations with specific complications of sickle cell anemia
Subphenotype Gene Genetic association study Reference
Stroke VCAM1/G1238C Candidate gene 188
VCAM1/T1594C Candidate gene 97
IL4R/S503P Candidate gene 97
TNFA/G-308S Candidate gene 97
TNF-α/−308G>A allele Candidate gene 21
LDLR/Ncol +/− Candidate gene 97
ADRB2/Q/27E Candidate gene 97
AGT/AG repeats Candidate gene 186
HLA genes Candidate gene 182

HLA genes Candidate gene 98

GOLGRI and ENPPI Genome-wide association and 72
whole-exome sequencing
ENPPI Candidate gene 22
α-Thalassemia, ADYC9, ANXA, TEK, Candidate gene 71
TGFBR3
Osteonecrosis MTHFR/C677T Candidate gene 227
IL-1β (−511C>T and +3954C>T Candidate gene 201
BMP6 Candidate gene 15
BMP6 Candidate gene 196
Acute chest syndrome NOS3/T-786C Candidate gene 175
NOS1/AAT repeats Candidate gene 183
COMMD7 Gene-centric association 76
HMOX1 Candidate gene 19
Bilirubin levels and gallstones UGT1A/promoter repeats Candidate gene 148
UGT1A/promoter repeats Candidate gene 70
UGT1A/promoter repeats Candidate gene 200
UGT1A/promoter repeats Genome-wide association and 124
candidate gene
Priapism KL Candidate gene 139
KL Candidate gene 66
Pulmonary hypertension TGF-β/BMP pathway genes Candidate gene 10
IL-1β (−511C>T and +3954C>T Candidate gene 201
MAPK8 A allele Candidate gene 226
eNOS intron 4 VNTR polymorphism Candidate gene 187
Leg ulcers KL, TEK, TGF-β/BMP pathway genes Candidate gene 138
IL-6 (−597G>A and −174G>C) genes Candidate gene 201
Bacteremia TGF-β/BMP pathway genes Candidate gene 4
Renal dysfunction APOL1 Candidate gene 11
APOL1, α-thalassemia, BCL11A Candidate gene 170
α-Thalassemia, APOL1, HMOX1 Candidate gene 78
Acute pain studies have not been included owing to the poor quality of the data.

signaling pathway (ACVRL1, BMPR2, and BMP6) (10) and others previously implicated in pri-
mary idiopathic pulmonary hypertension (116). A more recent multicenter study (226) considered
the hypoxic response to be contributory to pulmonary hypertension. A SNP (rs10857560) as-
sociated with reduced expression of MAPK8, which encodes a mitogen-activated protein kinase
important for apoptosis, T cell differentiation, and inflammatory responses, correlated with pul-
monary hypertension.
A gene-centric association study of ACS using individuals from the US Cooperative Study of
Sickle Cell Disease found an association with rs6141803. This SNP is located 8.2 kb upstream
of COMMD7, a gene highly expressed in the lung that interacts with NF-κB signaling (76).
Another candidate gene is heme oxygenase 1 (HMOX1), which produces the protein HO-1, the
rate-limiting enzyme in the catabolism of heme, which might attenuate the severity of acute pain
and hemolysis. Bean et al. (19) investigated a highly polymorphic (GT)n dinucleotide repeat in
the HMOX1 promoter and showed that children with two shorter alleles had lower rates of ACS.
Studies have suggested that the incidence of bacterial infections among patients with SCA might
be modulated by polymorphisms in MBL2 at the HLA locus, MPO (encoding myeloperoxidase),
DARC, and the TGF-β/BMP pathway (4, 48, 136, 185).
Renal dysfunction, as measured by proteinuria or estimated glomerular filtration rate, correlates
with the severity of hemolysis (50). Hence, it is also not surprising that co-occurrence of α-
thalassemia is protective against albuminuria (50, 135). The MYH9-APOL1 locus, an important
genetic risk factor for end-stage renal failure in non-SCA populations of African ancestry (79), is
also associated with nephropathy in patients with SCA (11, 78, 170). The true association is broadly
considered to be with APOL1, owing both to the stronger statistical association with that gene
and to the lack of identification of causal functional variants in MYH9. The original association
with MYH9 has been attributed to the strong linkage disequilibrium between MYH9 and APOL1.
It must be said that, in general, the lack of replication in independent studies means that many
of the published genetic associations remain somewhat questionable. Contributing factors include
underpowered studies, a poor definition of phenotypes, and failure to control for confounders such
as population stratification and admixture, issues that are not unique to SCA. Of the published
genetic associations, beyond α-thalassemia and those specific to HbF, only two are strongly con-
vincing: the association between UGT1A1 and bilirubin levels and predisposition to gallstones and
the association between the MYH9-APOL1 locus and sickle nephropathy, which are compelling
because they have been replicated in different SCA cohorts and confirmed in populations without
SCA.
THE MANAGEMENT AND OUTCOME OF SICKLE CELL ANEMIA

IN HIGH-INCOME COUNTRIES
A major evidence-based review of the management of SCA was recently undertaken by the US
National Institutes of Health and published in 2014 (132, 223). A striking feature of that report
was that many recommendations are based on levels of evidence that are less than high, reflecting
the widespread paucity of data from randomized controlled trials. For many years, the corner-
stones of treatment for the majority of patients affected by SCA have been the prevention of
bacterial infections and the management of complications such as pain and end-organ damage. As
stated above, without specific interventions, the incidence of severe and fatal bacterial infections
is extremely high, particularly in the early years of life (31). However, early mortality from severe
bacterial infections has now been all but eliminated by the widespread implementation of prophy-
laxis with penicillin during the 1970s and 1980s and by the subsequent introduction of specific
vaccines for both S. pneumoniae and H. influenzae. Nevertheless, recent reports suggest that these

interventions do not completely eliminate the risk and that the incidence of invasive infections with
S. pneumoniae, particularly those caused by strains that are not covered by the standard pneumo-
coccal vaccines, remains unacceptably high (119, 134). Although rates of invasive pneumonococcal
disease fell by 65% in US children with SCD following the introduction of the PCV7 vaccine (120),
a recent study from Minnesota reported an incidence that is still 18 times that reported among
children without SCA in the postvaccine era (119). Such studies bring into question the practice
of discontinuing penicillin at 5 years of age, particularly given the low level of evidence on which
this practice is based (69, 132, 223). Parvovirus B19 infection, the cause of a mild flu-like illness
(the so-called fifth disease) in children without SCA, can have serious consequences in patients
with SCA. The most common manifestation is a transient but potentially fatal erythrocyte aplasia
that can lead to acute severe anemia (174), but infections can also contribute to the development
of ACS and nephrotic syndrome (162). As a consequence, for some years there has been interest
in the development of a parvovirus B19 vaccine for use in children with SCA. A leading candidate
currently in preclinical development, a Saccharomyces cerevisiae–derived virus-like particle-based
vaccine, was immunogenic in both wild-type mice (43) and a mouse model for SCA (150).
ESTABLISHED TREATMENTS FOR SICKLE CELL ANEMIA

For many years, specific treatments for the majority of patients with SCA have been limited to the
provision of analgesics during painful crises and blood transfusions for particular indications, in-
cluding episodes of ACS or severe acute anemia (132, 223). Two additional treatments—long-term
transfusion therapy with iron chelation and hydroxyurea—have been reserved largely for patients
with specific complications. However, informed by the growing appreciation of the pathophysi-
ological processes that underlie the complications of SCA, an increasing number of newer drugs
and treatments are now being developed, some of which could potentially prevent or reverse the
course of SCA.
Hydroxyurea
Until very recently, hydroxyurea was the only disease-modifying drug ever to be licensed for the
treatment of SCA by the US Food and Drug Administration. Hydroxyurea was originally used
as a chemotherapeutic agent in the management of malignancies, but its potential benefits as a
treatment for SCA were first recognized more than 30 years ago (157). Hydroxyurea is a potent
inhibitor of ribonucleotide reductase, a key enzyme in DNA synthesis (65), and this appears to be
key to its numerous therapeutic effects. Hydroxyurea has many of the attributes of an ideal drug
for SCA because it has multiple mechanisms of action that affect various points on the pathophys-
iological pathway described above. When administered once daily at the correct dose (typically
15–30 mg/kg, as determined by titration against hematological response indices), hydroxyurea
leads to therapeutic reductions in neutrophils, reticulocytes, platelet counts, and inflammatory
cytokines, all of which have been implicated in the process of vasoocclusion. Finally, hydroxy-
urea commonly (but not universally) results in the therapeutic induction of HbF, the beneficial
effects of which were discussed in detail above. Together, these mechanisms lead to lower levels
of hemolysis (a process implicated in endovascular injury), a consequent increase in steady-state
hemoglobin, and improved rheology of erythrocytes and neutrophils through reduced vascular
cytoadherence (210). Nevertheless, although these protean benefits of hydroxyurea have been
documented through multiple clinical trials conducted in both adults and children, its introduc-
tion into routine treatment has been surprisingly slow, and uptake in some countries (including
well-resourced ones) remains relatively low.

Chronic Transfusion Therapy

Chronic transfusion therapy has long been shown to reduce the risk of strokes in children who
are at increased risk on the basis of TCD monitoring (3). In the Stroke Prevention Trial in Sickle
Cell Anemia (STOP), transfusion therapy was associated with a 92% reduction in the risk of overt
stroke among children with TCD velocities of ≥200 cm/s and also reduced the risk of further
strokes in children with existing silent infarcts (53). However, the discontinuation of transfusion
therapy is associated with a high rate of reversion to abnormal TCD velocities and a high risk of
further strokes (111, 144). The Stroke with Transfusions Changing to Hydroxyurea (SWiTCH)
trial showed that continued transfusions with chelation therapy were superior to hydroxyurea
with phlebotomy in the management of children with an existing stroke and iron overload (212),
but the recently completed Transcranial Doppler with Transfusions Changing to Hydroxyurea
(TWiTCH) trial asked whether hydroxyurea could be used as an alternative approach to primary
stroke prevention in high-risk children. Because it is currently the standard of care, all children
were first established on transfusion therapy; however, TWiTCH showed that, in children who
have been transfused for at least one year, switching to hydroxyurea was as effective as continuing
transfusions in terms of TCD velocities (211).
Blood transfusion remains an essential element in the management of patients with SCA, and
indications for blood transfusion are expanding (59), but it is not without risk. Common com-
plications include secondary iron overload and hemolytic transfusion reactions caused by the
development of red blood cell antigen alloimmunization, and transmission of infections remains a
significant issue in many countries (46). The most feared complication is alloimmunization to red
blood cell antigens, which is relatively more common in patients with SCA and can lead to difficul-
ties in sourcing appropriately matched blood products, delayed transfusion reactions, significant
morbidities, and even fatalities in some cases (52, 204). Clinician awareness of delayed transfusion
reactions is poor because symptoms mimic acute vasoocclusive pain and immunohematology find-
ings are often negative. One possible reason for the relatively high incidence of alloimmunization
observed in the SCA population is the mismatch in the red blood cell antigens expressed in patients
of African descent and donors of primarily northern European descent (224). However, despite
ethnic matching, a significant proportion of SCA patients still became alloimmunized (47). Molec-
ular analyses of the RH genes in SCA patients and African American donors revealed remarkable
RH allelic diversity in this population and mismatch between serologic Rh phenotype and RHD or
RHCE genotype owing to variant RH alleles in 87% of individuals (47). As a prevention, many blood
transfusion centers have adopted extended red cell phenotyping, including ABO, Rh, Kell, Kidd,
Duffy, and S and s antigens, and some centers have also adopted molecular genotyping for red blood
cell phenotype prediction using microarray chips (e.g., the PreciseType HEA BeadChip assay).
NEW TREATMENTS FOR SICKLE CELL ANEMIA

As stated above, until very recently, the management of SCA was largely reactive. Complications,
particularly painful crises, have been managed as they occur, and preventive treatments have
been limited largely to hydroxyurea and blood transfusion. In the last two decades, however, the
situation has changed dramatically with the development of new drugs and treatments for specific
types of crisis and of other drugs that are aimed at preventing or reversing the development
of complications altogether. To some extent, this reflects the growing realization that even in
patients with a seemingly mild phenotype, ongoing chronic hemolytic anemia is a pathological
driver of insidious end-organ damage. The recent surge in new drugs has also been helped by the
designation of SCA as an orphan disease (a disease that affects fewer than 200,000 people in the

United States), with the result that drugs that are developed for its treatment benefit from the
Orphan Drug Act of 1983, which allows for significant financial incentives. The most significant
new drugs for the prevention and treatment of SCA are summarized in Table 3 and discussed in
further detail below.
The Prevention of Polymerization

As discussed above, the polymerization of deoxy-HbS is the root cause of the downstream conse-
quences of SCA. The development of a drug that prevents or mitigates this polymerization could
therefore be transformative. Several approaches have been considered, as summarized in a recent
comprehensive review (64). The best established is the induction of HbF synthesis, which holds
particular promise because there is plentiful evidence of efficacy from clinical and epidemiolog-
ical studies. As discussed above, the onset of symptoms among children with SCA is delayed for
several months, until HbF is replaced by HbS. Moreover, the proportion of HbF beyond that
point is one of few factors that correlates closely with outcome. Finally, it is believed that the
induction of HbF is a major element in the effectiveness of hydroxyurea therapy. Alternative ap-
proaches to HbF induction are under investigation, and several drugs have entered clinical trials.
Advances in our understanding of the molecular mechanisms regulating the switch from fetal to
adult globin-gene expression have led to the generation of new agents that fall into two main
groups: those that affect chromatin regulators (such as decitabine on DNA methylation and his-
tone deacetylase inhibitors) and those that affect DNA-binding transcription factors. BCL11A is
a potent repressor of HbF but has important roles in neuronal development and B cell function.
Lineage-specific, microRNA-mediated short hairpin RNA suppression of BCL11A in erythroid
cells led to stable long-term engraftment of gene-modified cells and increases in HbF (33). Dis-
section of the BCL11A erythroid-specific enhancer down to a small region in intron 2 of the gene
offers the possibility of disrupting this region and specifically targeting erythroid function using
genome-editing technology such as zinc-finger nucleases or CRISPR/Cas9 (37, 205). It might also
be possible to derepress γ-globin expression by forcing interaction of the β-locus control region
with the γ-globin gene using a synthetic DNA-binding protein (32, 56). This forced chromoso-
mal looping approach has the advantage that βS -globin expression is reduced concomitantly with
increased γ-globin expression.
In addition to agents aimed at inducing HbF production, drugs designed to inhibit polymer-
ization directly are under development. In theory, it should be possible to prevent polymerization
through the use of small molecules designed to block the sites at which the α- and βS -globin
chains bind during this process, and although no candidates have yet been identified, this approach
remains the target of active research (64). 5-Hydroxymethyl-furfural (Aes-103) is a naturally oc-
curring aldehyde that binds to the N-terminal valine residue of the α-globin chain of HbS and
results in a dose-dependent increase in its oxygen affinity (140). Effective at preventing polymer-
ization in vitro, Aes-103 was well tolerated and apparently safe in a phase 1 dose-escalation trial
conducted in adults (140). Although the final analysis is not yet complete, early results from a phase
2 trial (NCT01987908) show no obvious benefit of Aes-103 over placebo, and as a result, the trial
was terminated early. However, the study was designed to address dose and safety as opposed to
efficacy, with three-quarters of participants randomized to the active drug; further trials will
therefore be needed to determine its utility. A second drug has since been developed that acts
through the same mechanism but with higher specificity and at lower concentrations (123, 142).
GBT440 is a polyaromatic aldehyde that was safe in early phase trials and was associated with a
dose-dependent increase in hemoglobin, decreased reticulocytes, irreversibly sickled cell counts,
and serum bilirubin concentrations, consistent with reduced levels of early hemolysis. Multicenter

Table 3 New therapeutic agents for the prevention and treatment of complications
GG19CH06_Williams
Therapeutic target Mechanism Example(s) Route Status Prevention Treatment Comments Reference(s)
HbS polymerization
ARI
Blocking of High-affinity None Oral? Theoretical Currently theoretical 64
132
intermolecular small molecules
contact sites
Modification of Increase in O2 5-Hydroxymethyl- Oral Phase 2 Links to N terminus 140
Williams
hemoglobin structure affinity furfural (Aes-103)
28 July 2018
of α-globin
·
GBT440 Oral Phase 2 Activity similar to 63, 140, 142
Thein
5-hydroxymethyl-
12:37
furfural (Aes-103)
but higher affinity
Prevention of red Blocking of Senicapoc Oral Phase 3 Blocks the Gardos 13, 14
blood cell Gardos channel channel to prevent
dehydration dehydration; phase
3 trial −ve
Induction of HbF Dilution of red Butyrates, Oral Phase 2 Sodium dimethyl 164
blood cells decitabine, and butyrate (HQT-
(HbS) tetrahydrouridine 1001) clinically
unimpressive
Delivery of carbon Blocking of Sanguinate IV Phase 2 A bovine PEGylated 125; trial
monoxide polymerization hemoglobin product NCT02411708
Cytoadhesion
Reversal of Reduction of L-Glutamine Oral Licensed Glutamine depletion 128, 217
metabolite depletion cytoadherence contributes to
of sickled red membrane damage
blood cells
Adrenaline-induced Adrenaline Propranolol Oral Phase 1/2 Results awaited 51
erythrocyte adhesion inhibitor
Neutrophil and Anti- IV IV Phase 1/2 Results awaited Trial
monocyte adhesion inflammatory immunoglobulin NCT01783691
Simvastatin Oral Phase 2 Reduced
cytoadhesion of
white blood cells
(Continued)
Table 3 (Continued)
Therapeutic target Mechanism Example(s) Route Status Prevention Treatment Comments Reference(s)
GG19CH06_Williams
P-selectin inhibitors Blocking of adhesion Crizanlizumab IV Phase 3 Monoclonal 12

of activated antibody to
ARI
erythrocytes, P-selectin; phase 2

neutrophils, and study + ve
platelets
Pan-selectin Blocking of adhesion Rivipansel IV Phase 3 Synthetic small 190
28 July 2018
inhibitors of activated (GMI-1070) molecule; phase 2

erythrocytes, trial + ve
neutrophils, and
12:37
platelets
Non-ionic surfactant Blocking of adhesion Poloxamer 188 IV Phase 3 PEO-PPO-PEO 145, 168
of activated and compounds
erythrocytes, vepoloxamer nonspecifically
neutrophils, and improve rheology
platelets
Heparin-like Sevuparin IV Phase 2 Inhibition of red Trial
molecules cell cytoadhesion NCT02515838
via P-selectin
N-acetyl cysteine Antioxidant
Platelets
Reduced platelet Platelet inhibitor Prasugrel and Phase 2/3 Prasugrel trials −ve 91; ticagrelor
activation and ticagrelor in phase 3; phase 2 study
aggregate formation study of ticagrelor NCT02482298
complete, results
awaited
Coagulation
Anticoagulants Reduction of the Low-molecular- IV Phase 2 Results awaited Trials
activation of weight heparins NCT01419977
coagulation in (dalteparin and and
www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes

vasoocclusive crises unfractionated NCT02098993
heparin)
133
Factor Xa Oral Phase 3 Results awaited Trials
inhibitors NCT02072668
(rivaroxaban and
and apixaban) NCT02179177
Abbreviations: HbF/S, hemoglobin F/S; IV, intravenous; PEO, poly(ethylene oxide); PPO, poly(propylene oxide); + ve, clinical trial results showed a benefit; −ve, clinical trial results showed no
benefit.
phase 3 clinical studies of GBT440 are now being launched in both adults (NCT03036813) and
children (NCT02850406), the results of which are awaited with considerable interest.
While two further approaches to the prevention of sickling showed early promise, neither
proved effective in clinical trials. The kinetics of HbS polymerization are exquisitely sensitive to
HbS concentration, and in theory, reducing the intracellular concentration of HbS by as little
as 10% could be beneficial. Senicapoc (ICA-17043) counters the potassium leak and dehydration
that are characteristic of the cellular pathology of SCA by targeting the Gardos channel. While
early results were promising in terms of markers of reduced hemolysis and improved erythrocyte
survival (14), a phase 3 trial proved negative in terms of efficacy in the prevention of vasoocclusive
crises (13). Finally, Sanguinate, a bovine PEGylated hemoglobin product, was designed to block
the polymerization of HbS through the delivery of carbon monoxide, a potent antisickling agent.
Sanguinate is administered intravenously, and its potential benefits are limited to the treatment of
vasoocclusive events. A phase 1 trial found sufficient evidence of safety (125) to allow its progression
to a phase 2 trial (NCT02411708).
Preventing and Reversing Vasoocclusion

Several drugs that have been designed to target the general area of vasoocclusion are in various
stages of development, some of which have shown considerable promise in clinical trials. The
recently described involvement of endothelial adhesion molecules has led to the development of
several specific inhibitors, with a particular focus on the selectins. Crizanlizumab, an intravenously
administered monoclonal antibody that targets P-selectin, has shown efficacy in both the preven-
tion and reversal of the sequestration process in in vitro models, and results from early trials in
humans have been promising. Most recently, a placebo-controlled trial [the Study to Assess Safety
and Impact of SelG1 with or without Hydroxyurea Therapy in Sickle Cell Disease Patients With
Pain Crises (SUSTAIN)] of high-dose crizanlizumab (5.0 mg/kg), administered intravenously 14
times over a period of 52 weeks, found that patients treated with the active drug had a 45.3%
lower rate of crises (P = 0.01) (12). Crizanlizumab had an additive effect in patients who were re-
ceiving concomitant hydroxyurea. Among patients receiving hydroxyurea, those given high-dose
crizanlizumab had an annual crisis rate that was 32.1% lower than those given a placebo.
STEM CELL TRANSPLANTATION AND GENE THERAPY

Allogeneic hematopoietic stem cell transplantation has become an increasingly acceptable treat-
ment option for SCA. An analysis of 1,000 HLA-identical sibling donor transplantations con-
ducted between 1986 and 2013 has revealed excellent outcomes, with both children and adults
demonstrating 93% (95% confidence interval 91.1–94.6%) overall survival (82). Modifications to
the intensity of conditioning have expanded allogeneic hematopoietic stem cell transplantation as
a treatment option for adult patients with preexisting organ dysfunction, who would have been
otherwise ineligible for transplantation using standard myeloablative conditioning (99). However,
less than 14% of patients with SCA have HLA-matched siblings as potential donors. Alternative
sources of half-matched (haplo-identical) family donors have therefore been actively explored to
make hematopoietic stem cell transplantation available to more patients (30).
Gene therapy using genetically modified patient-derived (i.e., autologous) hematopoietic stem
cells would avoid the risk of graft-versus-host disease and could therefore be applicable to many
more patients (40, 84). Several gene therapy approaches are being explored. One approach is
that of gene addition using lentiviral vectors (β-like globin transgenes, short hairpin RNA for
erythroid-specific expression, and lentiviral expression of a ZF-LDB1 fusion protein). The most

advanced of these is the antisickling β-globin vector containing the HbAT87Q mutation (Bluebird
Bio), which was first tested in a patient with transfusion-dependent HbE/β-thalassemia (41). The
first case of SCA treated by this approach reported therapeutic levels (>50%) of antisickling β-
hemoglobin, no evidence of insertional mutagenesis, an absence of crises, and the correction of
disease hallmarks (166). Interim results from a phase 1/2 study using this same vector revealed
therapeutic hemoglobin HbAT87Q expression in all seven patients with SCD. These data should re-
assure patients regarding the short-term safety of gene therapy, although long-term safety remains
an open question.
The other gene therapy approach is gene editing. SCA presents an ideal opportunity for gene
editing because of its SNP-based etiology, but the method involves nuclease-induced homology-
directed repair, which is highly inefficient. Much more promising are approaches that cause the
disruption by nonhomologous end joining of the erythroid-specific enhancer in the γ-globin

repressor (BCL11A); however, this method is error prone. The creation of mutations in the HBB
complex to simulate natural hereditary persistence of fetal hemoglobin variants by gene editing is
another approach under active investigation (193, 215).
CURRENT PERSPECTIVES IN LOW-INCOME SETTINGS

While many of the exciting developments that we have outlined above hold great promise for
improving the lives of patients born with SCA in high-income countries, the current reality is very
different for the vast majority of patients who are born with SCA in low-income regions, where,
until very recently, the condition has been almost completely neglected as a health priority. No
country within these regions has yet established a national screening program, and as a result, most
patients come to medical attention only when they present with complications. As many as 90%
therefore die, usually undiagnosed, during the first 5 years of life (87). As a consequence, in many
parts of Africa, SCA is probably responsible for an astonishing 5–16% of total mortality under age
5 (127). Only a small fraction of all research into SCA is conducted in the regions most affected,
meaning that, to a great extent, the guidelines that are in place are not grounded in the local context.
The last few years, however, have seen several welcome developments. The importance of SCA
has been formally recognized by multiple international organizations, including the United Na-
tions and the World Health Organization (197, 220). Moreover, bodies like the National Heart,
Lung, and Blood Institute of the US National Institutes of Health are starting to appreciate the
importance of better funding through the establishment of specific funding calls (133). Further col-
laboration among governments, scientists, and international funding organizations is badly needed
if the plight of the majority of the world’s SCA population is to be improved in the years ahead.
CONCLUSIONS
In the century since SCA was first formally recognized in the scientific literature, it has been
at the forefront of numerous scientific discoveries. To a large extent, the translation of these
discoveries into new drugs and treatments has been much less impressive, partly because of the
low number and marginalized status of those affected in resource-rich regions. In recent years,
however, improvements in survival in conjunction with a growing appreciation of the true burden
of this condition more globally have led to a greater focus on the translation of scientific knowledge
into new therapies that could soon transform the outlook for affected patients regardless of where
they live. Perhaps most exciting are gene therapy approaches that now hold the promise of complete
cure, although a great deal more will need to be learned about the balance of risk and benefit
before such treatments can be rolled out to any but the most severely affected. In the meantime,

a concerted effort to discover and develop new drugs aimed at preventing the complications of
SCA is still justified, because such approaches will probably come with fewer risks at a lower cost
and will be accessible to a broader range of patients.
SUMMARY POINTS
1. Sickle cell anemia (SCA) has been at the vanguard of human genetic discovery since its
recognition by Linus Pauling as the first example of a molecular disease in 1949.
2. The identification of genetic variants of hemoglobin F as important phenotypic modifiers
provided early proof of principle that genome-wide association studies could contribute
meaningfully to therapeutic developments.
3. Genetic and genomic developments are now translating to the development of new
therapeutics that may radically reduce disease morbidity.
4. A cure for this chronic and debilitating disease is now within reach through stem cell
transplantation, gene therapy, and gene editing.
5. Much more remains to be done for the vast majority of affected patients who are born in
low-income regions within Africa and India.
FUTURE ISSUES
1. Although it seems likely that curative therapies such as gene editing and stem cell trans-
plantation will gain traction in the years ahead, a range of issues, including safety, cost,
and timing, mean that new disease-modifying drugs will be needed for the foreseeable
future.
2. SCA has evolved from a life-threatening disease of childhood into a chronic disease in
adults, with many unmet needs.
3. Morbidity and mortality remain significant problems. The life expectancy of someone
with SCA still lags behind that of others without SCA by more than 20 years.
4. An inequity of treatment exists not only between high- and low-income countries, but
also within well-resourced countries.
5. There is a need to develop a network of experts who could serve as resources for
community-based providers.
6. Prediction of disease severity remains an issue; we need more, and better, biomarkers
and genetic markers to allow us to focus the potentially toxic treatments (hydroxyurea,
stem cell transplant, and gene therapy) on those patients with high-risk disease.
7. It is now clear that all patients with SCA, however mild their symptoms appear to be, are
continuously undergoing some degree of chronic end-organ deterioration. It is therefore
crucial that new drugs target the mechanisms that lead to this end-organ damage.
8. As new drugs and treatments are developed, it will be essential to find ways to make them
available to patients in low-income countries who stand to benefit most.

NOTE ADDED IN PROOF

Until recently, it has been accepted that the βS -globin mutation arose at least four times, expanding
through diverse ethnic groups but limited to five geographically distinct haplotypes: Senegal,
Benin, Central African Republic/Bantu, Cameroon, and Arab–Indian. The selective force involved
in the expansion of the gene was most likely Plasmodium falciparum malaria; the time of the gene
frequency increase was likely to have been during the expansion of agriculture, approximately
4,000 or more years ago in India and approximately 3,000 years ago in Africa. However, a recent
paper by Shriner & Rotimi (176a) used whole-genome sequence data and molecular typing to
classify 20 β-globin haplotypes in linkage disequilibrium with rs334, and presented compelling
evidence that rs334 arose once, on the HAP1 haplotype, from which the other haplotypes were
then derived. Modeling of balancing selection of the heterozygote advantage estimated that the
sickle mutation arose approximately 7,300 years ago in West Africa.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
T.N.W. is funded by the Wellcome Trust through Senior Research Fellowship 202800. S.L.T. is
supported by the intramural program of the National Heart, Lung, and Blood Institute, National
Institutes of Health.
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GG19CH06_Williams ARI 28 July 2018 12:37

Annual Review of Genomics and Human Genetics

Sickle Cell Anemia and Its

Thomas N. Williams1,2 and Swee Lay Thein3

Annu. Rev. Genom. Hum. Genet. 2018. 19:113–47 Keywords

The Annual Review of Genomics and Human Genetics Abstract

THE MOLECULAR BASIS OF SICKLE CELL DISEASE

THE ORIGINS, GLOBAL BURDEN, AND DISTRIBUTION OF SICKLE

114 Williams · Thein

1963 1978 1995 2010 2016

1846 1949 1985 2000 2015 2018

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 115

HbS allele frequency (%)

116 Williams · Thein

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 117

HbS Polymerized HbS

Oxygenated RBC Deoxygenated RBC Dehydrated sRBC

Hemolytic Neutrophil adhesion

Heme Neutrophil NO binding

Endothelial cell VWF Selectin

118 Williams · Thein

CLINICAL COURSE AND COMPLICATIONS

Acute Manifestations of Sickle Cell Anemia

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 119

Table 1 Commonly recognized complications of sickle cell anemia

catheterization postcapillary secondary to left

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes

Hemorrhagic and Cardiac: pulmonary

Sickle hepatopathy Sickle nephropathy

Bone infarction Leg ulceration

122 Williams · Thein

Chronic Complications of Sickle Cell Anemia

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 123

as measured by two-dimensional echocardiography, are suggestive of pulmonary hypertension, and

FACTORS DETERMINING OUTCOME

124 Williams · Thein

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 125

126 Williams · Thein

HLA genes Candidate gene 182

HLA genes Candidate gene 98

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 127

THE MANAGEMENT AND OUTCOME OF SICKLE CELL ANEMIA

128 Williams · Thein

ESTABLISHED TREATMENTS FOR SICKLE CELL ANEMIA

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 129

Chronic Transfusion Therapy

NEW TREATMENTS FOR SICKLE CELL ANEMIA

130 Williams · Thein

The Prevention of Polymerization

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 131

Blocking of High-affinity None Oral? Theoretical Currently theoretical 64

P-selectin inhibitors Blocking of adhesion Crizanlizumab IV Phase 3 Monoclonal 12

erythrocytes, P-selectin; phase 2

inhibitors of activated (GMI-1070) molecule; phase 2

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes

Preventing and Reversing Vasoocclusion

STEM CELL TRANSPLANTATION AND GENE THERAPY

134 Williams · Thein

disruption by nonhomologous end joining of the erythroid-specific enhancer in the γ-globin

CURRENT PERSPECTIVES IN LOW-INCOME SETTINGS

www.annualreviews.org • Sickle Cell Anemia and Its Phenotypes 135

136 Williams · Thein

NOTE ADDED IN PROOF