Hydroxyurea treatment in β-thalassemia patients: To respond or not to respond?

ArticleinAnnals of Hematology 92(3) · January 2013with127 Reads
Impact Factor: 2.63 · DOI: 10.1007/s00277-012-1671-3 · Source: PubMed
  • 20.08 · University of Social Welfare and Rehabilitation Sciences
Abstract

Hydroxyurea (HU) is a drug that induces fetal hemoglobin production. As a result, HU is widely used to treat β-thalassemia (β-thal) patients. However, the response of these patients to HU varies. Some β-thal patients respond favorably to treatment while others do not respond at all. HU has a number of side-effects and therefore its targeted prescription is beneficial. Hence, identifying the genetic determinants which lead to the differential HU response is important. This review summarizes recent findings which have shed light on this topic. Special emphasis is given to the mechanisms and genetic loci which may govern these differences. These findings have helped identify several single nucleotide polymorphisms which associate with the response to HU in both β-thal and sickle cell disease patients.

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Available from: Mehdi Banan
REVIEW ARTICLE
Hydroxyurea treatment in β-thalassemia patients:
to respond or not to respond?
Mehdi Banan
Received: 24 December 2012 /Accepted: 29 December 2012 /Published online: 15 January 2013
#
Springer-Verlag Berlin Heidelberg 2013
Abstract Hydroxyurea (HU) is a drug that induces fetal
hemoglobin production. As a result, HU is widely used to
treat β-thalassemia (β-thal) patients. However, the response
of these patients to HU varies. Some β-thal p atients respond
favorably to treatment while others do not respond at all.
HU has a number of side-effects and therefore its targeted
prescription is beneficial. Hence, identifying the genetic
determinants which lead to the differential HU response is
important. This review summarizes recent findings which
have shed light on this topic. Sp ecial emphasis is given to
the mecha nisms and geneti c loci which may govern these
differences. These findings have helped identify several
single nucleotide polymorphisms which associate with the
response to HU in both β-thal and sickle cell disease
patients.
Keywords β-thalassemia
.
Hydroxyurea
.
Pharmacogenomics
.
HbF
Abbreviations
HU Hydroxyurea
β-thal β-thalassemia
SNP Single nucleotide polymorphism
SCD Sickle cell disease
Hb Hemoglobin
HbF Fetal hemoglobin
HbA Adult hemoglobin
β-TI β-thalassemia intermedia
β-TM β-thalassemia major
HPFH Hereditary persistence of fetal hemoglobin
GR Good responder
MR Minor responder
NR Nonresponder
GWAS Genome-wide association study
QTL Quantitative trait loci
Molecular basis of β-thalassemia
The hemoglobin (Hb) molecule is composed of two α-globin
chains and two β-like globin chains [1]. Expression of the
human α-globin gene, located on chromosome 16, begins
shortly after life and persists throughout adulthood [24]. In
contrast, the β-like globin genes are expressed in a spatially
and temporally restricted manner (Fig. 1). The human β-like
globin genes are composed of five structural genes (ε, Gγ, Aγ,
δ,andβ) located on the β-locus of chromosome 11 [24]. The
ε-globin gene is expressed during the first month postgesta-
tion in the yolk sac. Afterwards, ε-globin is silenced and
expression of the fetal γ-globin genes (Gγ and Aγ)commen-
ces in the fetal liver and spleen to produce the fetal hemoglo-
bin or HbF (α
2
γ
2
). A second switch occurs shortly after birth.
At this time, the γ-globin genes are silenced and expression of
the adult δ-andβ-globin genes begins in the bone marrow. At
this stage, the predominant hemoglobin is the adult hemoglo-
bin or HbA (α
2
β
2
)[24].
β-thalassemia (β-thal) is an autosomal recess ive disorder
that is caused by mutations in the β-globin gene [5, 6 ].
Persons heterozygous for β-globin mutations are merely
carriers (termed β -thal trait). However, patients homozy-
gous for β-g lobin mutations develop anemia and iron
M. Banan (*)
Genetics Research Center, University of Social Welfare
and Rehabilitation Sciences, Evin, Daneshjoo Blvd.,
Koodakyar St,
Tehran, Iran
e-mail: mbbanan@yahoo.com
Ann Hematol (2013) 92:289299
DOI 10.1007/s00277-012-1671-3
Page 1
overload, followed by complications such as bone deformi-
ties, splenomegaly, and growth retardation [5].
To date, over 200 mutations in the β-globingenehavebeen
identified [7, 8]. These mutations may reside in the β-globin
promoter, untranslated region (UTRs), exons, or splice sites.
As a result of these mutations, β-globin transcription, transla-
tion, or RNA processing becomes impaired. Subsequently, β-
globin expression becomes abolished (β°), severly reduced
(β
+
), or slightly diminished (β
++
). Deletions in the β-globin
gene, on the other hand, are relatively infrequent [9]. These
deletions range from 290 bp to >80 kb. The larger deletions
may result in removal of the δ-andβ-globin genes. A com-
plete list of the β-globin mutations and deletions can be found
in the HbVar database (http://globin.cse.psu.edu/)[10].
In β-thal patients, reduction in β-globin expression causes
an imbalance in the α-toβ-globin chain ratios in the red blood
cells (RBCs) [5, 9]. This imbalance, in essence, leads to the
pathophysiology of the disease. The excess α-globin chains
precipitate and form inclusion bodies, damaging and destroying
the RBCs through apoptosis. Depending on the extent of the α/
β chain imbalance, patients may develop a mild or severe
anemia leading to conditions referred to as β-thal intermedia
(β-TI) a nd β-thal major (β-TM), respectively.
Reactivation of HbF as a therapeutic strategy
Reactivation of the fetal γ-globin genes in adults may serve
as a therapeutic strategy for the treatmen t of β-thal patients
[11]. An early indication for this pr emise came from a
condition known as the hereditary persistence of fetal he-
moglobin (HPFH). Persons with HPFH have high HbF
levels (1030 %) even as adults [2, 3, 7]. In one variety,
HPFH is caused by deletions in the β-locus (termed
deletion -type HPFH). These deletio ns, which r ange f rom
7 kb (e.g., Corfu deletion) to >80 kb (e.g., Black deletions),
result in the reactivation of γ-globin expression.
Two mechanisms may account for the upregulation of γ-
globin expression by HPFH deletions. Certain HPFH dele-
tions may result in juxtaposition of a distant enhancer next
to the γ-globin genes. Experiments using transgenic mice
carrying the human β-locus with HPFH-like deletions pro-
vide evidence for this model [1214]. Alternatively, these
deletions may lead to the removal of a γ-globin silencer
element. Recently, it has been determined that in persons
with HPFH, the common truncated region contains binding
sites for the γ-globin repressor, BCL11A [15, 16].
Certain H PFH deletions (e.g., HPFH-1 and HPFH-2)
begin just 3 of the Aγ gene and lead to the removal of the
δ- and β-globin genes. In spite of this, individuals bearing
these deletions are clinically normal [3, 7]. Persons homo-
zygous for these truncations have high Hb levels (1518 g/
dL) and their Hb is entirely comprised of HbF. Moreover,
patients wh o are co mpound heterozygous for β-globin
mutations and these HPFH deletions show clinically mild
symptoms [ 7 ]. These observatio ns imply that reactivation of
γ-globin expression to levels matching the HPFH deletions
may serve as a therapeutic strategy for treating β-thal
patients.
In part as a result of these observations, a number of
chemicals have been identified to reactivate HbF expression
in patients with Hb disorders [1720]. Prominent examples
include sodium butyrate (a histone deacetylase inhibitor), 5-
azacytidine (a methyl-transferase inhibitor), and hydroxy-
urea (HU; a DNA repli cation inhibitor). However, HU is
currently the only drug that is prescribed for the treatment of
anemia in β-thal patients.
Response of β-thal patients to HU
HU (or hydroxycarbamide) is a chemotherapeutic agent,
which was initially used to treat patients with myeloprol if-
erative disorders [17, 18, 21]. In the 1980s, it was discov-
ered that HU c an induce HbF expr ession in sickle cell
disease (SCD) patients. The observation followed experi-
ments aimed at understanding the mechanism of HbF in-
duction by the nucleoside analogue, 5-azacytidine (5-Aza)
[21]. At the time, it was debated as to whether 5-Aza was
inducing HbF production in SCD patients through DNA
hypomethylation or via inhibition of DNA synthesis. HU
was a well-known ribonucleotide reductase inhibitor (an
Fig. 1 The β-like globin genes are expressed in a developmentally
restricted manner. Schematic of the human β-globin-like genes (ε, Gγ,
Aγ, δ, and β) located on chromosome 11 is depicted (LCR locus control
region). Also shown is the temporal expression of these genes. The
graph is adapted from [4]
290 Ann Hematol (2013) 92:289299
Page 2
enzyme that is required for the generation of deoxyribonu-
cleotides and DNA replication) with no known DNA methyl-
transferase inhibitor activity [22](Fig.2). Thus, to provide
insight, the effect of HU on HbF production was tested in
monkeys [21]. Results of these experiments showed that HU
can induce HbF expression in baboons [21]. More impor-
tantly, ensuing clinical trials demonstrated that HU treatment
can induce HbF expression in SCD patients [2326]. HU also
reduced the incidence of painful crises and alleviated the acute
chest syndrome in these patients [2327]. As a result, HU has
been approved by the US Food and Drug Administration
(FDA) for the treatment of SCD patients.
At present, HU is also widely used to treat β-thal patients
[28 41]. In a subset of these patients, HU treatment leads to the
improvement of hematological parameters (HbF%, Hb, mean
corpuscular volume, and mean corpuscular hemoglobin). These
responder patients can generally be divided into two groups:
good responders (GR) and minor responders (MR) [32, 33, 36,
41]. In transfusion-dependent patients, the donor blood is
mixed with that of the recipient and thus perturbs the Hb values.
Therefore in these p atients, HU response is measured throu gh
increased blood transfusion intervals (note: transfusion is gen-
erally initiated when Hb <8 g/dL). As such, the transfusion-
dependent GR patients (either β-TM or β-TI) show a signifi-
cant increase in their blood transfusion intervals post-HU treat-
ment (>6 months) [32, 36, 41]. The transfusion-independent
GR patients (β-TI), on the other hand, show a significant
increase in their Hb levels (2 g/dL) posttreatment [33].
However , not all β-thal patients respond favorably to the
drug. While some transfusion-dependent MR patients shift
from regular to sporadic blood transfusions posttreatment,
others show a modest 23 fold increase in their blood transfu-
sion intervals [33, 36, 41]. Furthermore, some transfusion-
independent MR patients merely exhibit a 1 g/dL increase in
their Hb levels posttreatment [33]. More importantly, approx-
imately 2030 % of β-thal patients do not respond to HU
treatment at all (termed nonresponders (NR)) [28
41].
HU has a number of benefits, which has resulted in its
widespread use. In particular, HU can be taken orally, is
inexpensive, and is considered to be safe [26, 42, 43]. In the
long-term, however, HU may not be as safe as is generally
perceived. Several reports suggest that HU treatment in
SCD patients may lead to leukemia, impaired spermatogen-
esis, and leg ulcers [44, 45]. HU may also produce somatic
mutations in children with SCD [46]. In addition, HU treat-
ment may cause adverse side-effects in β-thal patients.
These side-effects include headaches, hyperpi gmentation,
nausea, and dizziness [40].
Because of the potential adverse effects and lack of
response in a subse t of patie nts, ta rgeted presc r iption of
HU is preferable. Targeted prescription, however, requires
an understanding of the reasons behind the differential re-
sponse of patients to the drug.
Explanations for the differential HU response
Two mechanisms may account for differences in the response
of β-thal patients to HU. In one possibility, the erythroid cells
of responders and NR may react differently to HU treatment.
The erythroid cells of NR may, for instance, upregulate HbF
less vigorously due to a deficiency in the γ-globin induction
pathways. Or else, the cells of NR may be more susceptible to
the cytotoxic effects of HU. Here, this has been termed the
differential susceptibility model. Alternatively, HU treatment
may augment HbF production in both the responder and NR
patients. The HU response, however, may only become man-
ifested in patients who have higher cellular HbF levels. Here,
this has been labeled the differential baseline HbF model.
Several lines of evidence support the differential baseline
HbF model. Recent findings suggest that HU treatment can
induce HbF production from the erythroid progenitor cells
of both the responder and NR β-thal patients (15 GRs and
12 NRs). The baseline HbF levels, however, seem to be
significantly higher (i.e., 20-fold) in erythroid progenitors of
the responders [47].
In addition, several cohort studies suggest that the pre-
treatment peripheral blood HbF levels (Hb and HbF%) are
higher in the responder patients [33, 39, 41 ]. In one study
involving 79 Indian β-thal patients (41 β-TM and 38 β-TI),
for example, the baseline HbF levels in the β -TI patients
were as follows: GR (Hb, 7.6 g/dL; HbF, 51.8 %)>NR (Hb,
6.3 g/dL; HbF, 25.9 %) [39]. In another cohort of 37 Indian
β-TI patients, the pretreatment HbF levels were as follows:
GR (Hb, 6.5 g/dL; HbF, 67.0 %)>NR (Hb, 6.5 g/dL; HbF,
40.9 %) [33]. Of note, transfusion-dependent patients have
also been included in these cohorts. Unfortunately, Hb levels
Fig. 2 The chemical structure of HU is shown. The regions of HU
involved in ribonucleotide reductase inhibition (blue-shaded area) and
nitric oxide generation (green-shaded area) are highlighted. The figure
is adapted from [26]
Ann Hematol (2013) 92:289299 291
Page 3
in transfusion-dependent patients are influenced by the donor
blood and may not be completely reliable. Nevertheless, some
of the reported differences between the responder and NR β-
thal patients are too high to overlook [39].
On the other hand, there is also evidence to support the
differentia l susceptibility model. Several cohort studies suggest
that HbF and HbF producing cell (termed F cells) levels are
increased posttreatment only in the responder patients. In one
study, for instance, Hb and HbF% levels were significantly
increased only in the GR patients (Hb, 1.5 g/dL; HbF,
35 %) [39]. Similar results were obtained in another study
(Hb, 2.6 g/dL; HbF, 9%)[33]. Furthermore, a significant
increase (>30 %) in F cell levels of GR patients has also been
reported after HU treatment [39].
In further support of this model, expression microarray
experiments show that the erythroid progenitors of responders,
in contrast to that of NR, have an activated stress response
program (see section below). High-expression levels of several
such genes (specifically ARG1, ARG2,andBCLX
L
)maypro-
tect the erythroid cells of responders from cell stress and
apoptosis, thus allowing them to expand in the presence of
HU [47]. As a result, the erythroid cells of responders may
become immune to the cytotoxic effects of HU.
In summary, HU response in β-thal patients may be
determined by differences in (1) pretreatment HbF levels
of the erythroid cells and (2) the response of erythroid cells
to HU treatment.
The mechanism of HbF induction by HU
As noted above, the erythroid cells of responders and NR
may react differently to HU treatment (differential suscepti-
bility model). Here, a summary of the mechanisms by which
HU may induce γ-globin expression is provided. A better
understanding of these mechanisms may offer further in-
sight into the differential response of β-thal patients to HU.
Two mechanisms have been proposed to explain HbF
induction following HU treatment [48]. In one, HU may
promote stress erythropoiesis to increase the number of F
cells. Alternatively, HU may activate signaling pathways
which lead to γ-globin upregulation. These two course s
of action are not mutually exclusive [48]. In particular,
HU may upregulate the expression of both γ-globin and
genes which promote stress erythropoiesis. In concert,
these events could lead to the production of high HbF
levels posttreatment.
Stress erythropoiesis and HbF induction
It has been proposed that inhibition of DNA synthesis by
HU may lead to stress erythropoiesis, similar to what occurs
during conditions of low oxygen [49]. In particular, it is
hypothesized that HU may block DNA synthesis in the
rapidly dividing erythroid precursors which produce HbA.
This may, in turn, lead to a selective advantage for expan-
sion of the less mature HbF producing F cells [50, 51]. As a
result, this so-called stress erythropoiesis would lead to
the induction of HbF production. In suppor t of this notion, a
reduction in burst forming unit-erythroid cell level s and an
increase in F cell levels have been reported following HU
treatment in SCD and β-thal patients [39, 52, 53].
Further support for this model comes from several ex-
pression profiling studies. In one study, using reticulocytes
from children with SCD, HU treatment downregulated the
expression levels of genes invol ved in translation, ribosome
assembly, and chromosome organization [54]. These results
suggest that HU may alter the kinetics of erythropoiesis by
inhibiting protein (rather than DNA) synthesis. Another
report has shown that GATA-1 levels in the erythroid pro-
genitor cells of healthy donors decrease following HU treat-
ment [55].ReducedGATA-1expressioncandelaythe
maturation of erythroblasts [56]. This delay may, in turn,
alter the kinetics of erythropoiesis to favor HbF production.
In addition, HU may induce the expression of a number of
apoptosis-related genes (e.g., DR5, caspase-3,andBCL6)in
the erythroid precursor cells of both healthy adults and β-
thal patients [47, 56]. As noted above, selective activation of
several such genes (e.g., BCLX
L
and BCL6) may protect the
erythroid precursors of NR from apoptosis [47].
The signaling model
Two cell types have been widely utilized to investigate the
HU/γ-globin induction pathways. One model system is the
K562 erythroleukemia cell line [57]. K562 cells express the
γ-globin gene and more importantly, γ-globin expression in
these cells is induced following HU treatment [58, 59]. The
more biologically relevant cells are the erythroid progenitors
of healthy persons and patients with Hb disorders, which
also upregulate γ-globin expression in response to HU
treatment [60, 61]. Using these cell types, a number of the
HU/γ-globin induction pathways have been deciphered
(Fig. 3).
In one pathway, induction of γ-globin may occur
through the generation of nitric oxide (NO). Once taken
orally, HU can rapidly spread from the intestine to blood
cells through facilitated uptake by solute carrier trans-
porters [62]. HU can then react with heme to produce
NO [63]. The gener ated NO can then nitrosylate (and
activate) the soluble guanylate cyclases to produce
cGMP [64, 65]. Subsequently, the generated cGMPs
can lead to the induction of γ-globin expression [66].
The downstream activator(s) of this pathway have not
been identified. However, possibilities include the AP-1
(c-fos/jun) and the Sp1 transcription factors [67].
292 Ann Hematol (2013) 92:289299
Page 4
In addition, cGMP can downregulate phosphodiesterase
3 expression to activate the cAMP pathway [68]. Activation
of the cAMP pathway results in upregulation of γ-globin
expression in erythroid progenitor cells [69]. However, the
opposite effect is seen in K562 cells, a phenomenon that has
been linked to induction of the already high MYB levels in
these cells (MYB may act as a γ-globin repressor) [70].
HU can also upregulate γ-globin expression through the
p38 MAPK/CREB1 pathway. Firstly, both p38 MAPK and
CREB1 seem to be important in maintaining steady-state γ-
globin expression levels [71]. Furthermore, HU treatment (like
sodium butyrate) can lead to the phosphorylation of p38
MAPK in K562 cells [7274]. We have further established that
in K562 cells, CREB1 is phosphorylated following HU treat-
ment, and its knockdown by RNA interference blocks γ-globin
induction [75, 76]. Collectively, these results underscore the
importance of this signaling pathway in γ-globin induction.
In addition, specific miRNAs may be involved in the
upregulation of γ-globin expression by HU. In particular,
the expression levels of two miRNAs (mi R-26b and miR-
151-3p) seem to be increased in the reticulocytes of SCD
patients following HU-treatment [77] . Furthermore, this
upregulation has been associated with increased HbF levels.
Whether these miRNAs play a direct role in γ-globin upre-
gulation, however, has yet to be determined.
QTLs that affect baseline HbF levels
As discussed above, genomic loci which affect baseline HbF
levels may also influence the response of β-thal patients to
HU (differential baseline HbF model). Genome-wide asso-
ciation studies (GWAS) have identified three quantitative
trait loci (QTLs) that affect baseline HbF levels in healthy
persons and in patients with Hb disorders (Table 1). Several
single nucleotide polymorphisms (SNPs) in these loci may
account for 2050 % of the HbF variance [78, 79]. Notably,
minor alleles of these SNPs also associate with a milder
anemia in β-thal patients.
The XmnI polymorphism
A well-known HbF QTL i s the XmnI polymorphism
(rs7482144), a C T SNP at position -158 of the Gγ promoter
[80]. Early reports and recent GWA studies show that presence of
the XmnI T allele correlates with higher HbF levels in β-thal and
SCD patients [
8183]. In addition, a large twin study suggests
that this SNP can influence F cell levels in healthy adults [84].
The XmnI polymorphism has also been correlated with
reduced disease severity in β-thal patients. In particular, a
report shows that frequency of the XmnI T allele is higher in
the French β-TI patients compared with patients having β-
TM [85, 86]. In support, we have observed a significant
correlation between the XmnI T/T genotype and β-TI in a
cohort of >300 Iranian patients [87]. Despite these associa-
tion data, no function for the XmnI SNP has been estab-
lished. Therefore, it has been postulated that linked elements
in the β-locus rather than the XmnI polymorphism itself may
affect γ-globin expression [88].
BCL11A
Another HbF QTL lies in the BCL11A gene. BCL11A is a
developmental repressor of the γ-globin gene [89, 90]. In
particular, knockdown of BCL11A in human erythroid pro-
genitor cells can result in a significant increase in γ-globin
expression [89]. In addition, BCL11A knockout induces γ-
globin expression in human β-locus transgenic mice [91, 92].
Several GWAS and replication studies have associated
SNPs in intron 2 of the BCL11A gene (e.g., rs11886868,
rs4671393, and rs766432) with HbF levels in healthy per-
sons and in patients with hemoglobinopathies [83, 9396].
Furthermore, a GWAS has correlated one of these SNPs
(rs766432) with F cell levels in SCD patients [97]. The
minor alleles of these SNPs have also been correlated with
a milder disease phenotype (i.e., β-TI) in French, Italian,
and Iranian patients [8587, 93, 98
]. Preliminary data by
Orkin and colleagues suggest that this BCL11A intronic
region may contain an erythroid-specific enhancer [99].
HBS1L-MYB
A third HbF QTL has been located between the HBS1L and
MYB genes. Several SNPs in this intergenic region (e.g.,
Fig. 3 Summary of the signaling pathways leading to γ-globin induc-
tion in erythroid progenitors and K562 cells is depicted. HU can induce
γ-globin expression by increasing nitric oxide (NO) and cAMP levels
or through phosphorylation of p38 MAPK and CREB1. sGC soluble
guanylate cyclase, pDE3 phosphodiesterase 3
Ann Hematol (2013) 92:289299 293
Page 5
rs9399137 and rs4895441) have been associated with HbF
and F cell levels in healthy persons and in patients with
hemoglobinopathies [83, 93, 94, 100, 101]. The MYB onco-
gene can act as a γ-globin repressor in erythroid cells [102,
103]. Thus, the HBS1L-MYB intergenic region is likely in-
volved in regulation of MYB expression. In support of this
notion, this region contains erythroid-specific DNase I hyper-
sensitive sites and histone acetylation patterns [104]. Further-
more, a number of sites within this region have enhancer
activity and form long-range interactions with the MYB pro-
moter during mouse erythroid cell development [104, 105].
Markers that predict the HU response
Predictive markers in SCD patients
Several SNPs have been identified which associate with the
HU response in SCD patients. In a cohort of 386 adult SCD
patients, a number of SNPs in the promoter and 5UTR of the
SAR gene associated with the HU response [106]. SAR is a
gene that is involved in protein trafficking [59]. SAR expres-
sion is induced by HU and its over-expression leads to γ-
globin upregulation in CD34
+
cells [106]. It has therefore been
suggested that SAR may increase the transport of γ-globin
transcription factor precursors from the ER to the Golgi [59].
Furthermore in a cohort of 137 SCD patients, an associ-
ation study of 29 candidate genes has shown a correlation
between the HU response and SNPs in several stress re-
sponse genes (e.g., FLT1, NOS1 , TOX, ARG1, and ARG2)
[107]. Interestingly, a recent study shows that ARG1 and
ARG2 are upregulated in the erythroid cells of β-thal Re-
sponder patients after HU treatment [46]. However, the
associations of these SNPs with the response to HU have
not been investigated in β-thal patients.
In a study involving 93 SCD children, minor alleles of
two BCL11A SNPs (rs4671393 and rs1427407) associated
with reduced pretreatment BCL11A levels and increased
baseline HbF% levels in the patients reticulocytes [54].
In another prospective association study of 174 SCD
children under HU treatment, the XmnI polymorphism and
several SNPs in the BCL11A gene correlated with baseline
HbF% levels [108]. In additio n, SN Ps in th e AR G1 and
ARG2 genes associated with increased HbF% levels post-
treatment. However none of the 70 selected SNPs, which
were in the ribonucleotide reductase, HU transporter, and
HbF modifier genes, correlated with HbF% levels at the
maximum tolerated dose of HU [108].
Predictive markers in β-thal patients
An association has been established between the XmnIpoly-
morphism with the response to HU in β-thal patients (Table 2).
Several studies have shown an association between the XmnI
T/T genotype with a robust HU response and the XmnIC/C
genotype with a lack of response [31, 32, 36, 39, 41]. How-
ever, other studies have failed to detect such a correlation [33].
These studies have used different inclusion criteria (e.g., num-
ber of β-TM and β-TI patients) and have not utilized a
uniform definition of HU response (Table 2
). Some of these
inclusion criteria (e.g., β-thal type and co-inheritance of α-
thal) may affect the response to HU [31, 38, 39, 109]. There-
fore, a direct comparison of these results is not possible.
In addition, we have shown an association between minor
alleles of two linked BCL11A SNPs (rs766432 and
rs4671393) with the response to HU in transf usion-
dependent β-thal patients (Table 2)[41]. By using both the
XmnI T/T and the BCL11A rs7664 32 markers, we were able
to predict the HU response in >85 % of the β-thal patients.
Interestingly, a recent repor t shows that HU treatment
alone does not trans-activate reporter gene expression from
mouse erythroid leukemia (MEL) cells stably transfected
with a dual-reporter modified human β-globin locus con-
struct (MEL
A
γRedβEGFP). However, HU treatment pre-
ceded by BCL11A knockdown led to a synergistic
upregulation of reporter g ene (
A
γRed) expression [110].
These results provide insight into how BCL11A expression
may influence the HU response in β-thal patients [54].
Future directions
Insights into the mechanisms of HU-mediated γ-globin induc-
tion have led to the identification of several SNPs which
Table 1 Summary of the HbF QTLs which influence HbF and F cell levels is shown
Locus Chromosome Key SNPs Populations HbF/F cell Associated with β-TI References
β-globin 11 rs7482144 Healthy adults, SCD,
and β-thal
HbF and
F cells
Yes [8287]
BCL11A 2 rs766432, rs11886868,
and rs4671393
Healthy adults, SCD,
and β-thal
HbF and
F cells
Yes [83, 85, 9398]
HBS1L-MYB 6 rs9399137 and rs4895441 Healthy adults, SCD,
and β-thal
HbF and
F cells
Yes [83, 85, 9398]
Also indicated is the associ ation of these SNPs with β-TI
294 Ann Hematol (2013) 92:289299
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Table 2 Summary of association studies which have correlated candidate SNPs with the HU response in β-thal patients
Cohort
location
Cohort
size
Thal type Definition of HU response Number of responders Predictive marker(s) References
β-TM β-TI GR MR NR
Iran 45 β-TM=36
and β-TI=9
β-TM patients=becoming transfusion
independent after treatment; β-TI patients
=an increase in Hb levels by >1 g/dL
25/36
(70 %)
9/9
(100 %)
XmnI T/T associated with
a response
[31]
Iran 133 β-TM Good response (GR)=regular transfusion-
stransfusion independent; minor
response (MR) =regular transfusion-
s>6 months transfusion intervals; and
no response (NR)=no change in
transfusion requirements
81/133
(61 %)
31/133 (23 %) 21/133 (16 %) XmnI T/T associated with
a good response
[32]
India 37 β-TI GR=patients becoming transfusion
independent or Hb increases by >2 g/dL;
MR 50 % decrease in transfusion
requirements or Hb increases by
12 g/dL; and NR=no change in
transfusion requirements
17/26
(65 %)
9/26
(35 %)
11/37
(30 %)
XmnI showed no
correlation with
response
[33]
Algeria 54 β-TM=45
and β-TI=9
GR=decrease in annual transfusion
requirements by >70 %; MR=decrease
by 4070 %; and NR=no change in
transfusion requirements
28/54
(52 %)
9/54
(17 %)
17/54
(31 %)
XmnI C/C associated with
a worse response
[36
]
Israel 18 TM=11
and β-TI=7
For transfusion-dependent patients,
response=patients becoming transfusion
independent and for transfusion-
independent patients, response =
Hb increases by 2 g/dL
9/11
(82 %)
2/7
(29 %)
The XmnI T allele
associated with a
response in the β-TM
patients
[38]
India 79 β-TM=41
and β-TI=38
GR=patients becoming transfusion
independent; MR =50 % reduction in
transfusion requirements; and NR=no
change in transfusion requirements
22/79
(28 %)
19/81
(23 %)
38/81
(47 %)
The XmnI T/T genotype
associated with GR in
β-TI patients
[39]
Iran 81 β-TM=54
and β-TI=27
GR=transfusion dependenttransfusion
independent; MR =2-fold increase in
transfusion intervals; and NR =no
change in transfusion intervals
37/81
(46 %)
24/81
(30 %)
20/81
(25 %)
XmnI T/T and BCL11A
rs766432 C and
rs4671393 A alleles
associated with response
[41]
Ann Hematol (2013) 92:289299 295
Page 7
associatewiththeHUresponseinβ-thal patients. In several
retrospective association studies, the XmnIandBCL11A SNPs
have been correlated with the HU response in β-thal patients
[31 , 32, 36, 39, 41]. As a first step, it is necessary to verify the
predictive ability of these markers in prospective association
studies by using large cohorts from diff erent populations.
In addition, associations of the XmnIandBCL11A markers
with the HU response have not been established in SCD
patients [108]. Conversely, associations of SNPs in several
stress response genes, which correlate with the HU response
in SCD patients, have not been tested in β-thal patients [107].
Of special interest are the ARG1 and ARG2 genes, which also
show differential expression levels posttreatment in β-thal
responder and NR patients [47]. In order to form a unified
scheme of the HU response, associations of these markers
needs to be cross-checked between the two disease types.
Finally, it would be interesting to determine the entire set
of SNPs which correlate with the HU response in both β-
thal and SCD patients. Such GWA studies would require a
large number patients, which necessitates a collaborative
multicenter approach to the p roblem [83, 93, 94]. In all
likelihood, several such SNPs should fall in loci known to
affect γ-globin expres sion. However in the process, new
loci modulating the HU response may also be discovered.
Acknowledgments The author would like to thank Dr. Sjaak Phili-
psen (Erasmus Medical Center, Rotterdam, The Netherlands) and Dr.
Farzin Pourfarzad (Sanquin Blood Bank, Amsterdam Medical Center,
Amsterdam, The Netherlands) for their critical comments on the
manuscript.
Conflicts of interest The author declares that he has no conflicts of
interest.
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