thalassemia

1. Genetic association studies in ␤-hemoglobinopathies
Swee Lay Thein1
1Department of Haematological Medicine, King’s College Hospital National Health Service Foundation Trust,
King’s College London School of Medicine, London, United Kingdom
Characterization of the molecular basis of the ␤-thalassemias and sickle cell disease (SCD) clearly showed that
individuals with the same ␤-globin genotypes can have extremely diverse clinical severity. Two key modifiers, an innate
ability to produce fetal hemoglobin and coinheritance of ␣-thalassemia, both derived from family and population
studies, affect the pathophysiology of both disorders at the primary level. In the past 2 decades, scientific research had
applied genetic approaches to identify additional genetic modifiers. The review summarizes recent genetic studies and
key genetic modifiers identified and traces the story of fetal hemoglobin genetics, which has led to an emerging
network of globin gene regulation. The discoveries have provided insights on new targets for therapeutic intervention
and raise possibilities of developing fetal hemoglobin predictive diagnostics for predicting disease severity in the
newborn and for integration into prenatal diagnosis to better inform genetic counseling.
Introduction
Among the hemoglobinopathies, sickle cell disease (SCD) and
␤-thalassemia have the most impact on morbidity and mortality,
affecting millions worldwide.1 Both are prototypical Mendelian
single gene disorders affecting the ␤-globin (HBB) gene. Despite
the apparent genetic simplicity, both disorders display extreme
clinical heterogeneity.2,3 Major advances have been made in our
understanding of the molecular pathology, pathophysiology, and
treatment of these disorders, but predicting the clinical course
remains difficult. This is particularly so for SCD, which presents
challenges in genetic counseling and management. Identification of
the genetic modifiers could provide more precise estimates of
disease severity and defining the genetic variation within the
pathobiological pathways could provide clues and targets for
therapeutic intervention.
The 2 major modifiers, an innate ability to produce fetal hemoglobin
(HbF, ␣2␥2) and coinheritance of ␣-thalassemia, have been derived
from more than 50 years of extensive biochemical and pathophysi-
ological studies and were subsequently confirmed by genetic
studies. The mechanisms of the modifying effects of HbF and
␣-thalassemia in SCD and ␤-thalassemia could not be more
different at the molecular level,yet these genetic modifiers have a
large clinical effect due to their impact on disease pathophysiology
at the primary level. In addition, the genetic variants are common
and their contribution to disease burden is substantial.
Rationale for genetic studies and approaches used to
demonstrate genetic modifiers
HbF levels and coinheritance of ␣-thalassemia are unlikely to be the
only major modifiers because they do not completely explain the
clinical diversity in either disorder. In SCD, for example, analysis of
sibling pairs suggested a genetic contribution to stroke risk not
explained by HbF levels or coinheritance of ␣-thalassemia; a child
with sickle cell anemia (SCA) has an increased risk of stroke if they
have had siblings who have experienced an overt stroke. Studies
have also shown a concordance in response to hydroxyurea therapy
among siblings. Observations of clinical variability between identi-
cal twins (who have identical genetic make-up)4 and longitudinally
within the same individual highlight the important contribution of
environmental factors to the phenotypic variability. Environmental
factors such as physical activity, diet, and toxins can elicit changes
in the gene activity (altering epigenome or “software”) without
changing the DNA code (or “hard-drive”). Such epigenetic changes
may account for the discordant clinical phenotypes in identical
twins and contribute to the complex disease etiology in both
disorders.5 Due to the complex interaction of the multiple factors, a
genetic approach might be the most efficacious way of identifying
the modifier genes and provide a better understanding of the disease
mechanisms.
All 3 approaches that are generally used to unravel genetic modifiers
in human disorders have been applied to the ␤-hemoglobinopathies:
mouse models, family and epidemiological studies, and genetic
association studies. Although mouse models of ␤-thalassemia and
SCD2 have been very important in providing proof-of-principle for
globin gene regulation along with development and testing of
therapeutic compounds, no novel genetic modifiers have been
discovered using this approach. In part, this may be due to a lack of
mouse models that replicate the complications encountered in SCD
(eg, stroke). Insights into the 2 major genetic modifiers, HbF levels
and ␣-globin genotype, were derived from an understanding of the
disease pathophysiology and were subsequently validated by family
and population genetic association studies (Tables 1, 2). Association
of serum bilirubin levels and predisposition to gallstones with
polymorphisms in the promoter of the UGT1A1 gene (Gilbert
syndrome) have been consistently demonstrated in many studies in
patients with ␤-thalassemia and SCD of all ages and different
populations.6,7 A recent single study suggested that the MYH9-
APOL1 locus, an important genetic risk factor for renal failure in
non-SCD population of African ancestry, is also associated with
sickle cell nephropathy.8 These association studies were based on
candidate genes selected according to one’s understanding of the
disease pathophysiology or prompted by association with the same
phenotypes in non-SCD populations.9
Candidate gene association studies look for differences in the
frequencies of genetic variants in targeted genes between cases and
controls. If a variant is more common in cases than controls, then
association can be inferred. The more recent genome-wide genetic
HEMOGLOBINOPATHIES: FRESH IDEAS FOR MANAGEMENT
354 American Society of Hematology

2. association studies (GWAS) involve an unbiased scan of the whole
human genome and, by design, are more likely to reveal unsus-
pected interactions. GWAS will also confirm previous candidate
genes if the association is robust.10,11 A case in point is the
application of GWAS in the highly successful discovery of BCL11A
(an oncogene that hitherto was not known to have a role in
erythropoiesis) as a quantitative trait locus (QTL) controlling
HbF.10,12 GWAS also confirmed association of the other 2 loci,
Xmn1-HBG2 (rs782144) on chromosome 11p and HBS1L-MYB
(HMIP) on chromosome 6q, which were previously discovered
through candidate and genetic linkage studies, with HbF production.
Similarly, GWAS confirmed the association between bilirubin level
and UGT1A1 polymorphism in SCD.11
It has become clear from the genetic association studies of HbF and
other common diseases and traits that GWAS can work13 but that
sample size matters, that clearly defined and well harmonized
phenotypes are critical, that replication and collaboration (interdisci-
plinary in addition to increasing sample size) matters, that current
hypotheses regarding candidate genes and pathways may not matter
so much, and that several genes can influence more than one disease
or trait. It has also become evident that simpler phenotypes such as
HbF, which are reproducible, measurable, and disease related, are
much more robust and successful in genetic association studies than
clinical end points. Such intermediate end points or endophenotypes
are often quantitative traits and thus provide more power in genetic
strategies. For quantitative continuous traits, one could focus on
extremes of the trait as a strategy to reduce genotyping or sequencing
costs, as was successfully applied in the GWAS for HbF and F cells.10
The brain is one major site of morbidity in children with SCD.
Increased velocity in the middle cerebral artery as detected by
transcranial Doppler (TCD) screening is a biomarker of early
cerebrovascular disease. Studies have shown that chronic blood
transfusion therapy at this stage can prevent overt stroke. True
primary stroke prevention, however, should prevent vascular dam-
age before TCD velocity becomes abnormal. TCD velocity would
therefore be an extremely attractive endophenotype in studies for
detecting genetic variants associated with sickle vasculopathy and
stroke risks.
Table 1. Genetic modifiers of SCD
A. Primary at level of HbS polymerization and sickling
Modifier Mechanism
1. Sickle genotype; HbSS, HbSC, HbS␤-thalassemia Affects HbS polymerization
2. Coinheritance of HbF QTLs Hybrid tetramer (␣2␤S␥) does not take part in HbS polymerization and HbF dilutes
intracellular HbS concentration
3. Coinheritance of ␣-thalassemia ␣-thalassemia reduces intracellular HbS concentration, decreases HbS
polymerization and hemolysis
B. Secondary at level of subphenotypes and complications
Modifier Complication and mechanism
1. UGT1A1 promoter (TA)n polymorphism Hyperbilirubinemia and gallstones
2. MYH9–APOL1 locus Proteinuria and sickle cell nephropathy
3. ADYC9, ANXA2, TEK and TGFBR3 Ischemic stroke risk, mechanisms unclear
4. GOLGB1 and ENPP1 Ischemic stroke risk, mechanisms unclear
5. TGF␤-/SMAD /BMP pathway Multiple subphenotypes including osteonecrosis, acute chest syndrome, pulmonary
hypertension, leg ulceration, renal impairment, infection, priapism
6. KLOTHO (KL) Priapism, mechanisms unclear
7. NPRL3 on Chr16p Reduces hemolysis, ␣-thalassemia effect suggested
Most modifiers that affect SCD at the secondary level of subphenotypes and complications have not been replicated.
Table 2. Genetic modifiers of ␤-thalassemia
A. Primary at level of ␣: non-␣-globin chain imbalance
Modifier Mechanism
1. ␤-globin genotype (one or two, and severity of
␤-thalassemia alleles)
Directly affects output of ␤-globin and chain imbalance
2. ␣-globin genotype
␣-thalassemia Reduces chain imbalance and ␣-globin excess
Coinheritance of extra ␣-globin genes (␣␣␣/, ␣␣␣␣/,
or HBA cluster duplication)
Increases ␣-globin excess and chain imbalance
3. Co-inheritance of HbF QTLs, eg, SNPs in BCL11A,
HMIP, Xmn1-HBG2
Increased ␥ chains combine with excess ␣-reducing chain imbalance
4. Potential modifiers include variants in ubiquitin
proteolytic pathway
Promotes proteolysis of excess ␣-globin
5. ␣-hemoglobin stabilizing protein (AHSP) Chaperones excess ␣-globin (studies inconclusive)
B. Secondary at level of complications related to disease and therapy
Modifier Complication and mechanism
1. UGT1A1 promoter (TA)n polymorphisms Hyperbilirubinemia and gallstones
2. HFE–eg.H63D variants Iron loading due to increased GI absorption
3. Variants in VDR, COL1A1, COL1A2, TGFB1 genes Osteopenia and osteoporosis, modification of bone mass
4. Apolipoprotein (APOE) ⑀4 Cardiac disease, risk factor for left ventricular heart failure,
mechanism unknown
5. Glutathione-S-transferase M1 Increase risk of cardiac iron loading, mechanism unknown
Hematology 2013 355

3. Whole genome or exome sequencing using next-generation sequenc-
ing technology in combination with well-defined phenotypes offers
the possibility of identifying new genetic variants. GWAS in
combination with exome sequencing identified mutations in
GOLGB1 and ENPP1 with stroke protection in SCA.14 In this study,
overt stroke was the clinical marker, but these variants have yet to
be independently validated in a different population group.
Review of genetic modifiers in ␤-hemoglobinopathies
For both SCD and ␤-thalassemia, factors that affect the primary
event of the disease process will have a global effect on the disease
phenotype. These include the causative genotype, coexisting ␣-thala-
ssemia, and the innate ability to produce HbF.
SCD should be considered as both a qualitative and quantitative
genetic disorder in that it is caused by the presence of an abnormal
Hb variant (HbS, ␣2␤2
S, HBB glu6val, GAG ␤6 GTG), yet the
likelihood of HbS polymerization and sickling is highly dependent
on the intra-erythrocyte HbS concentration. Homozygosity for HbS
(SCD-SS) or SCA is the most common genotype; the other
causative genotypes include compound heterozygous states of HbS
with HbC (SCD-SC) or ␤-thalassemia variants (SCD-S␤0 thalasse-
mia and SCD S␤ϩ thalassemia).2 Individuals with SCD-SS or
SCD-S␤0 thalassemia, in whom the intracellular hemoglobin compo-
sition is almost all HbS, have the most severe disease, followed by
SCD-SC and SCD S␤ϩ thalassemia. Simple heterozygotes for HbS
(HbAS) who have 30% to 40% intracellular HbS, with HbA in
excess of HbS, are asymptomatic. Under exceptional circumstances,
however, such as intense physical activity and dehydration, the
consequent increased intracellular HbS concentration can induce
vasoocclusive pain.
Approximately one-third of SCD patients of African descent have
coexisting ␣-thalassemia due to the common deletional variant
(Ϫ␣3.7/).2 The majority are heterozygous (␣␣/␣Ϫ), with 3% to 5%
homozygous for the deletion (␣Ϫ/␣Ϫ). Coexisting ␣-thalassemia
reduces intracellular hemoglobin concentration, thereby reducing
HbS polymerization, reducing sickling, and decreasing hemolysis.
Although the coexisting ␣-thalassemias have a protective effect
against complications associated with severe hemolysis, such as
priapism, leg ulceration, and albuminuria, the increased hematocrit
and blood viscosity may account for the increase in other complica-
tions associated with microvascular occlusion, such as increased
acute pain, acute chest syndrome, osteonecrosis, and retinopathy.15
Coexisting ␣-thalassemia also reduces bilirubin with a quantitative
effect that is independent to that of the UGT1A1 promoter polymor-
phism.7 In Jamaicans, the absence of ␣-thalassemia and higher HbF
levels predict a benign disease. A subsequent analysis of 40 elderly
Jamaican patients (Ͼ 60 years of age), however, suggested that
coexisting ␣-thalassemia may promote longevity (51% had coexist-
ing ␣-thalassemia), but the trend failed to reach significance.16
Coinheritance of ␣-thalassemia blunts the response to hydroxyurea
therapy in SCD, which may be explained by its effect on HbF levels
and mean cell volume, 2 key parameters associated with hy-
droxyurea response.17 It is quite likely that ␣-thalassemia carriers
could have a poorer response to RBC membrane channel blockers
that aims to reduce sickling through preservation of cell hydration.
HbF reduces the propensity for HbS polymerization; the hybrid
tetramers (␣2␤2␥) inhibit HbS polymerization and the presence of
HbF dilutes down the intracellular HbS concentration. In view of its
impact at the primary level of disease pathology, one would expect
HbF levels to have a global beneficial effect. Indeed, HbF levels are
a major predictor of survival in SCD, and low levels of HbF have
been associated with increased risk of brain infarcts in young
children.18 At the subphenotype level, apart from the clear benefit of
high HbF levels with acute pain and leg ulceration, there are
disparities and less conclusive evidence for its effects on other
complications such as stroke, renal impairment, retinopathy, and
priapism.15 These disparate conclusions are likely to arise from
small sample sizes, even smaller numbers of end complications, and
disparities in ascertainment of phenotypes. The uneven beneficial
effect of HbF on sickle-related complications could also be related
to the different pathobiology of large and small vascular disease.
HbF levels vary considerably, from 1% to as high as 25% in
individuals with SCD-SS, and behave as a quantitative genetic trait
as in healthy individuals. Three QTLs, one in cis to the HBB gene
cluster represented by the Xmn1-HBG2 site (rs7482144), HBS1L-
MYB intergenic polymorphisms (HMIP) on chromosome 6q, and
BCL11A on chromosome 2p, are major regulators of common HbF
variation.10,12,19-22 Their effect on HbF levels varies with the
frequency of the HbF boosting (minor) alleles in different popula-
tion groups. In patients of African descent with SCD, the 3 loci
account for 16% to 20% of the variation in HbF levels with a
corresponding reduction in acute pain rate.23,24 The favorable effect
of HbF and its modifiers (BCL11A and Xmn1-HBG2) variants on
pain and TCD velocities is measurable even in young infants, as
demonstrated in the recently completed BABY HUG study.25
Several studies have investigated the association of candidate genes
implicated in pathophysiology of vasoocclusion and vasculopathy,
such as those encoding factors modifying inflammation, oxidant
injury, nitric oxide biology, vasoregulation, and cell adhesion, with
sickle-related complications, including stroke, priapism, leg ulcers,
avascular necrosis, renal disease, acute chest syndrome, gallstones,
and susceptibility to infection.15,26,27 The majority of the reported
associations have not been replicated or validated and are likely to
be false positives. Of the numerous association studies reported, the
most robust is the association between serum bilirubin levels and
predisposition to gallstones with the 6/7 or 7/7 (TA) repeats in the
UGT1A1 promoter.11 UGT1A1 polymorphism continued to have a
strong influence even during hydroxyurea therapy; in one study,
children with the 6/6 UGT1A1 genotype achieved normal bilirubin
levels, whereas children with 6/7 or 7/7 UGT1A1 genotypes did
not.6
Suggestions of a familial predisposition to stroke and its devastating
consequences has prompted numerous genetic and clinical associa-
tion studies on cerebrovascular complications involving either large
or small vessels.14,28,29 Of the 38 published single nucleotide
polymorphisms (SNPs) associated with stroke, the effects of
␣-thalassemia and SNPs in four genes (ADYC9, ANXA2, TEK, and
TGFBR3) could be replicated, although only nominally significant
association results were obtained.30 More recently, GWAS in
combination with whole-exome sequencing have identified muta-
tions in 2 genes, GOLGB1 and ENPP1, which are associated with
reduced stroke risk in pediatric patients, but, again, this needs
validation in independent studies.14 To overcome the small sample
size in end point complications, a study used a compound phenotype
that included one or more sickle-related complications (see review
by Thein27). Patients with complications had a higher frequency of
the platelet glycoprotein allele HPA-5B. In this small study, most of
the complications were osteonecrosis and only 4 individuals had
more than one complication. Because traditional methods are often
inadequate in association studies of complex traits, methods of
356 American Society of Hematology

4. evaluating multilocus data are promising alternatives. A GWAS was
applied to SCD based on a disease severity score that was derived
from a Bayesian network that integrates 25 different clinical and
laboratory variables.31 Several genes not known to be related to the
pathogenesis of SCD were identified, including KCN6 (a potassium
channel protein) and TNK5 (a gene encoding tankyrase-1, a possible
telomere length regulator). However, it is important to remember
that results from such analytical techniques are dependent on the
structure of a model assuming certain causalities and probabilities
of the different variables. More recently, GWAS identified an SNP
(rs7203560) in NPRL3 on chromosome 16p that was independently
associated with hemolysis (using a score derived by principle
component analysis).32 rs7203560 is in perfect linkage disequilib-
rium with SNPs within the ␣-globin gene regulatory elements
(HS-48, HS-40, and HS-33). rs720356 is also in linkage disequilib-
rium with ITFG3 that is associated with RBC mean cell volume and
mean cell hemoglobin in several GWAS of different population
groups.33 It is proposed that NPRL3 reduces hemolysis through an
independent thalassemic effect on the HBA1/HBA2 genes.
Hydroxyurea remains a major treatment option for SCD; its main
effect is mediated primarily through induction of HbF. Clinical
response to hydroxyurea therapy, however, is variable with variable
HbF response; a main determinant of response appears to be the
baseline HbF levels. Numerous genetic association studies on HbF
response to hydroxyurea have been reported, of which the associa-
tion with Xmn1-HBG2 seems to be the most robust. In the recently
completed BABY HUG trial, however, the HbF genetic modifiers
were not able to identify the “high” and “low” responders to
hydroxyurea.25 Table 1 summarizes the genetic modifiers of SCD.
In ␤-thalassemia, as in SCD, the causative genotype, coinheritance
of ␣-thalassemia and HbF, are the main modifiers of clinical
severity. These genetic factors have a major impact because they
affect the central mechanism underlying disease pathophysiology
(ie, the degree of globin chain imbalance and the excess of ␣-globin
chains). Almost 300 mutations (deletions and point mutations) that
down-regulate the ␤-gene have been described (http://globin.bx.
psu.edu/hbvar/menu.html). Functionally, the mutations range from
null mutations (␤0 thalassemia) that cause a complete absence of
␤-globin production to those that cause a minimal deficit: ␤ϩϩ
thalassemia (sometimes referred to as silent ␤-thalassemia because
of the minimally reduced RBC indices and normal HbA2 levels in
heterozygotes).34 The causative ␤-globin genotype has the largest
impact on disease severity. Homozygotes or compound heterozy-
gotes for 2 ␤0 thalassemia alleles cannot produce any HbA (␣2␤2);
such individuals generally have the most severe anemia and depend
on blood transfusions for life (thalassemia major).
In many populations in which ␤-thalassemia is prevalent, ␣-
thalassemia also occurs at a high frequency. Individuals who have
coinherited ␣-thalassemia have less redundant ␣-globin and tend to
have less severe anemia. The degree of amelioration depends on the
severity of the ␤-thalassemia alleles and the number of functional
␣-globin genes. At one extreme, patients who have coinherited the
equivalent of only one functioning ␣-globin (Ϫ/␣Ϫ, HbH genotype)
with homozygous ␤-thalassemia have less severe anemia (thalasse-
mia intermedia) if the ␤-thalassemia alleles are ␤ϩ, but a more
severe phenotype if they have ␤0 thalassemia.3
In heterozygous individuals with one ␤-thalassemia allele, coinheri-
tance of ␣-thalassemia normalizes the hypochromia and microcyto-
sis, but the elevated HbA2 levels remain unchanged. Increased
␣-globin production through coinheritance of extra ␣-globin genes
(triplicated, ␣␣␣/␣␣Ϫ or ␣␣␣/␣␣␣; quadruplicated, ␣␣␣␣/␣␣; or
duplication of the whole ␣-globin gene cluster, ␣␣/␣␣/␣␣) with
heterozygous ␤-thalassemia tips the globin chain imbalance further,
converting a typically clinically asymptomatic state to thalassemia
intermedia. The severity of anemia depends on the number of extra
␣-globin genes and the severity of the ␤-thalassemia alleles.
Increased HbF response as an ameliorating factor becomes most
evident in non-transfusion-dependent ␤0 thalassemia patients who
have a mild disease despite the complete absence of HbA.35 In this
case, the increased ␥-chains combine with the redundant ␣-globin to
form HbF (␣2␥2), reducing the chain imbalance. Again, the degree
of amelioration depends on the severity of ␤-thalassemia alleles (␤ϩ
or ␤0) and the number of coinherited HbF genetic determinants
(common QTLs including BCL11A, Xmn1-G␥ -158 C3T and SNPs
in HMIP on chromosome 6q.12,36-39
At the primary level of chain imbalance, the proteolytic capacity of
the erythroid precursors in catabolizing the excess ␣-globin has
often been suggested, but this effect has been difficult to define.
␣-hemoglobin–stabilizing protein, a molecular chaperone of ␣-glo-
bin, has also been suggested as another genetic modifier, but its
impact on disease severity has been inconclusive.40
Genetic variants could also modify the different complications of
␤-thalassemia that are directly related to the anemia and ineffective
erythropoiesis or to therapy, such as iron chelation treatment. The
secondary complications include jaundice and predisposition to
gallstones, osteopenia, and osteoporosis; iron overload; and cardiac
disease (see Table 2 for modifiers of ␤-thalassemia). The degree of
iron loading, bilirubin levels, and bone mass are quantitative genetic
traits and thus will be modified by genetic variants regulating
expression of these traits.
HbF concentration is a major modifier of disease severity for both
␤-thalassemia and SCD, prompting decades of study into its
regulation in adults.41 The study of HbF quantitative genetics and
molecular control of hemoglobin switching mirrors the rapidly
evolving focus of genomics research in humans, from the study of
natural mutants, gene mapping, and GWAS to applications of the
other “-omics” technologies such as comprehensive gene expression
profiling and DNA-protein interactions. Studies of the deletions in
the HBB cluster associated with heterocellular persistence of fetal
hemoglobin (HPFH)2 indicated a “repressive” element in the
intergenic region between the A␥-globin (HBG1) and ␦-globin
(HBD) genes and enhancer elements downstream of the HBB gene.
Point mutations associated with HPFH are clustered in regions of
the ␥-globin gene promoters that subsequently proved to be binding
sites for ubiquitous and erythroid-specific transcription factors.
Although the HPFH phenotypes could be reproduced in transgenic
mice carrying the human ␤-globin locus and the mutations shown to
alter in vitro binding patterns, unambiguous identification of
proteins directly involved in globin switching remained elusive. A
variety of transcription factors with roles in globin gene regulation,
such as GATA-1, KLF1, and SCL/TAL, were identified, but how
they regulate the switch from fetal to adult hemoglobin was still not
evident.41
Nonetheless, studies of these HPFH mutants and discovery of the
upstream regulatory elements (␤-locus control region) triggered a
series of experiments and led to the concept that switching of fetal to
adult hemoglobin involves 2 mechanisms: autonomous silencing of
Hematology 2013 357

5. the fetal globin genes and competitive access of the adult globin
gene to the upstream ␤-locus control region. However, it was
difficult to reconcile these concepts of Hb switching with the
common variable persistence of HbF in healthy adults and in
patients with SCD and ␤-thalassemia. In many cases, the inheri-
tance patterns of the modestly elevated HbF levels are not clear and
not linked to the HBB cluster on chromosome 11. An early example
is an extended family of Asian-Indian origin, in which segregation
analysis showed that the genetic determinant for HPFH was
inherited independently from the HBB cluster and eventually
mapped to chromosome 6q23.19 Observations of variable HbF with
different ␤S haplotypes first suggested that the HBB cluster is a
prime location for a HbF determinant, represented by the C3T
SNP (rs7482144) at position Ϫ158 of the G␥ promoter, known as
the Xmn1-HBG2 polymorphism.2 By early 2006, developments in
genetic tools and genotyping platforms expedited by the Interna-
tional Human Genome HapMap Project enabled the development of
GWAS that led to the identification of BCL11A on chromosome
2p16 as another locus modifying ␥-globin levels.10,12 The next clue
in the hemoglobin-switching puzzle again came from human
genetics provided by a Maltese family in which 10 of 27 members
had HPFH that segregated independently from the HBB cluster.42
Genetic studies followed by expression profiling of erythroid
progenitors identified KLF1 as the ␥-globin modifier in this family
in which members with HPFH were heterozygous for the nonsense
p.K288X mutation in KLF1. Soon, a steady stream of novel
mutations in KLF1 associated with increases in HbF were reported
in different populations (for review, see Borg et al43). The increases
in HbF occurred as a primary phenotype or in association with RBC
disorders such as congenital dyserythropoietic anemia.
Functional studies in primary human erythroid progenitors and
transgenic mice demonstrated that BCL11A acts as a repressor of
␥-globin; the silencing effect involves reconfiguration of the HBB
locus through interaction with GATA-1 and SOX6 that binds the
proximal ␥-globin promoters.44,45 Profiling of erythroid cells from
the Maltese family showed that KLF1 p.K288X carriers had reduced
BCL11A expression.42 KLF1 was also shown to be a direct activator
of BCL11A in human erythroid progenitor cells and in transgenic
mice.42,46 Previous knockout studies in mice had shown that KLF1
is essential for activation of ␤-globin expression, but the embryonic
and fetal globin genes remain fully activated in the absence of
KLF1.47 Collectively, these results have led to the proposal that
KLF1 has dual functions in the ␥-globin to ␤-globin switch: KLF1
activates HBB directly and also silences the ␥-globin gene indirectly
via activation of BCL11A.47
High-resolution genetic mapping and resequencing refined the 6q
QTL to variants in a 24-kb region between the HBS1L and MYB
gene, referred to as HMIP block 2.19 Work in primary human
erythroid progenitor cells48 and transgenic mice showed that this
region contains distal enhancers required for MYB activation.49,50
MYB, which encodes the c-MYB transcription factor, is a key
regulator of hematopoiesis and erythropoiesis. One suggestion of
how MYB modulates HbF levels is via its effects on erythroid
proliferation/differentiation balance. A low MYB environment
favors accelerated erythropoietic differentiation, leading to the
release of early erythroid progenitors that are still synthesizing
predominantly HbF.51 Another suggestion is that MYB represses
fetal globin gene expression via KLF1 activation of BCL11A.52 A
third suggestion is that MYB works via its effects on the TR2/TR4
pathway. MYB up-regulates the nuclear receptors TR2/TR4, which
are repressors of the ␥-globin gene. Therefore, down-regulation of
MYB leads to suppression of the TR2/TR4 pathway and up-
regulation of fetal globin gene expression. The emerging network of
HbF regulation also includes SOX6; chromatin-modeling factor
FOP; the NuRD complex; the orphan nuclear receptors TR2/TR4
(part of direct repeat erythroid definitive [DRED]); the protein
arginine methyltransferase PRMT5, involving DNA methylation;
and HDACs 1 and 2, which are epigenetic modifiers53 (Figure 1).
Regulators of the key TFs, such as microRNA-15a and 16-1, in
controlling MYB54,55 could also have a potential role in regulating
HbF levels.
Prospective targets for therapeutic induction of HbF
Identification of these genetic loci regulating HbF levels has
revealed cellular pathways providing insights for new therapeutic
targets for increasing HbF (Figure 1). The ideal target would be one
that mimics and enhances the effect of the genetic variants that have
been identified in modulating HbF levels, but does not affect other
biological pathways. Targets identified in the emerging network of
HbF regulation include MYB, KLF1, and BCL11A. Manipulating
MYB to achieve an adequate therapeutic window could be problem-
atic due to its pleiotropic role in hematopoiesis. KLF1 appears to be
key in the ␥-globin to ␤-globin switch; although KLF1 expression is
almost exclusively restricted to erythroid cells, there would be
difficulty in achieving specificity due to its broad array of erythroid
activity.47 Reducing BCL11A activity is an attractive approach;
proof-of-principle in HbF reactivation has already been demon-
strated in a variety of systems, including cell lines, primary human
erythroid cells, transgenics, and a mouse model of SCD with
reversal of characteristic organ damage.56 However, BCL11A has
essential roles in neuronal and B-lymphocyte development. Other
promising targets include the DNA-methylating and histone-
modifying enzymes. Proof-of-principle has already been provided
by the effective in vivo HbF induction properties of 5-azacytidine
and decitabine, both of which target the DNA methyltransferase
DNMT1.2 Another attractive target is modulating expression of the
orphan nuclear receptors TR2/TR4; one approach is inhibition of
lysine-specific demethylase 1 (LSD1) via tranylcypromine (TCP),
an antidepressive agent.57
From genetics to more precise risk prediction?
Although environmental factors are important in determining the
clinical outcome of both SCD and ␤-thalassemia, it is evident that
the genetic background of the affected individual imparts a substan-
tial contribution to the clinical severity and response to iron
chelators and hydroxyurea. Over the past 20 years, numerous
association studies have been published and roles for many modify-
ing factors and genes proposed, but the results are questionable
because of the lack of replication. Furthermore, the genetic evidence
generated about the phenotype has not been supported by functional
assays or relevant models. Even if the variants are verified, they are
likely to explain only a small fraction of the phenotype variation
and, therefore, have limited predictive value and clinical utility.
One exception is HbF genetics. HbF levels in adults are highly
heritable and controlled by 3 main loci, BCL11A, HBS1L-MYB
intergenic polymorphisms, and Xmn1-HBG2. In healthy Europeans,
the 3 loci explained more than 44% of the total variance in HbF
levels (BCL11A 15.1%, HMIP 19.4%, and Xmn1-HBG2 10.2%),10
whereas in African patients with SCA, the 3 loci contribute from
16% (Tanzanians)24 to 20% (African Americans)23 of the HbF
variance, with a corresponding reduction in frequency of acute
painful crises. The impact of the HbF QTLs on HbF response and
358 American Society of Hematology

6. reduction in blood transfusion requirements has also been demon-
strated in patients with ␤-thalassemia and HbE/␤-thalassemia.12,38
The relatively large fraction (20%-50%) of phenotypic variation in
HbF explained through a limited number of genetic loci is unique
for a disease-relevant trait in humans compared with other traits and
complex disorders, For example, in Crohn disease, 20% to 23% of
disease risk can be explained by 71 QTLs.13 HbF could be the first
human quantitative trait to be effectively predicted through genetic
assay systems and its value tested to inform clinical or pharmacoge-
netic management (eg, response to HbF-activating agents).
Based on the current state of knowledge, the 3 HbF QTLs should be
able to predict an individual’s ability to produce HbF within a
clinically meaningful confidence limit. Studies in Sardinia and
France showed that a combination of the Xmn1-HBG2 site, SNPs in
BCL11A and HMIP, together with ␣-globin genotypes can predict
75% to 80% of disease severity in ␤-thalassemia.37,39 In a cohort of
316 ␤0 thalassemia patients, delayed or absent transfusion needs
were correlated with status of the 3 HbF QTLs and the ␣-globin
genotype.58 In a recent study of patients with SCA, a panel of 14
SNPs comprising 6 from BCL11A and the rest from chromosome
11p15 encompassing the HBB gene complex and olfactory receptor
gene region explained 23.4% of HbF variability.59 The value of HbF
predictive genetics lies in the newborn and early childhood stage,
when it may be possible to combine this knowledge with other
laboratory and clinical parameters (eg, dactylitis in infants, leuko-
cyte count, hemoglobin level, and TCD velocity) to predict severe
disease outcomes such as stroke, frequent pain crises, and acute
chest syndrome in SCA,25 thus facilitating early implementation of
preventive therapy. In ␤-thalassemia, HbF predictive diagnostics
could be integrated with information on ␤-globin and ␣-globin
genotypes to better inform genetic counseling.
Conclusion
The generation of a personalized genetic risk score to inform
prognosis and guide therapeutics has been a major driver underlying
genetic association studies in numerous diseases and traits, includ-
ing the ␤-hemoglobinopathies.60 A challenge that faces all GWAS is
the translation of genetic risk variants into clinical utility. In this
regard, predictive HbF genetics has 2 advantages: (1) the clinical
impact of HbF is well documented and (2) a limited number of
QTLs account for a substantial proportion of the trait variance.
Already, identification of the loci regulating HBG production has
provided new insights and leads for therapeutic HbF reactivation. A
DNA assay-based diagnostic for the testing of one’s ability to
produce HbF could provide predictive information for those at risk
of early complications. The success of predictive testing will depend
on continued accurate identification of genetic and environmental
factors and its ultimate clinical utility.
Acknowledgments
The author thanks Claire Steward for help in preparation of the
manuscript and apologizes to the many investigators whose work
could not be cited due to space limitations.
Disclosures
Conflict of interest disclosure: The author declares no competing
financial interests. Off-label drug use: None disclosed.
Figure 1. Emerging network of ␥-globin regulators in adult life and prospective targets for therapeutic induction of HbF. Targets identified in
the emerging network of HbF regulation include the KLF1, BCL11A, and MYB genes and the TR2/TR4 nuclear receptors that associate with
corepressors DNA methyltransferase 1 (DNMT1) and lysine-specific demethylase 1 (LSD1). KLF1 has a dual role in the silencing of ␥-globin genes: it
activates BCL11A, a repressor of ␥-globin gene expression, and it also activates the ␤-globin gene directly. BCL11A interacts with the GATA1, FOG1,
and SOX6 erythroid transcription factors and with the NuRD deacetylase and remodeling complex to promote suppression of ␥-globin gene expression.
The nuclear receptors TR2/TR4 associate with corepressors DNMT1 and LSD1 as part of the DRED complex, a known repressor of embryonic and
fetal globin genes in adults. MYB contributes to HbF regulation via activation of KLF1 (which activates BCL11A), activation of the DRED complex, and
by modulating the number of F cells as part of its effect on erythroid differentiation kinetics and its pleiotropic effect on hematopoiesis.
Hematology 2013 359

7. Correspondence
Swee Lay Thein, King’s College London School of Medicine,
Molecular Haematology, The James Black Centre, 125 Coldharbour
Lane, London SE5 9NU, United Kingdom; Phone: 44-20-7848-5443/
5447; Fax: 44-20-7848-5444; e-mail: sl.thein@kcl.ac.uk.
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