Candidate sequence variants and fetal hemoglobin in children with sickle cell disease treated with hydroxyurea.
ABSTRACT Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin.
In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively), with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels.
These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease.
- SourceAvailable from: Vivien Sheehan[Show abstract] [Hide abstract]
ABSTRACT: Hydroxyurea has proven efficacy in children and adults with sickle cell anemia (SCA), but with considerable inter-individual variability in the amount of fetal hemoglobin (HbF) produced. Sibling and twin studies indicate that some of that drug response variation is heritable. To test the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated phenotype-genotype associations using whole exome sequencing of children with SCA treated prospectively with hydroxyurea to maximum tolerated dose (MTD). We analyzed 171 unrelated patients enrolled in two prospective clinical trials, all treated with dose escalation to MTD. We examined two MTD drug response phenotypes: HbF (final %HbF minus baseline %HbF), and final %HbF. Analyzing individual genetic variants, we identified multiple low frequency and common variants associated with HbF induction by hydroxyurea. A validation cohort of 130 pediatric sickle cell patients treated to MTD with hydroxyurea was genotyped for 13 non-synonymous variants with the strongest association with HbF response to hydroxyurea in the discovery cohort. A coding variant in Spalt-like transcription factor, or SALL2, was associated with higher final HbF in this second independent replication sample and SALL2 represents an outstanding novel candidate gene for further investigation. These findings may help focus future functional studies and provide new insights into the pharmacological HbF upregulation by hydroxyurea in patients with SCA.PLoS ONE 10/2014; 9(10):e110740. · 3.53 Impact Factor
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ABSTRACT: Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.PLoS ONE 09/2014; 9(9):e106924. · 3.53 Impact Factor
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ABSTRACT: Background Despite compelling evidence that hydroxyurea is safe and effective in sickle cell disease, it is prescribed sparingly due to several barriers like knowledge gaps in certain genotypes, apprehension about its safety and toxicity, and limited resources. We undertook this study to find out the efficacy and safety of HU in patients with HbSβ+-thalassemia with IVS1–5(G C) mutation. Procedure We registered 318 patients with HbSβ+-thalassemia with IVS1–5(G C) mutation. Of these, 203 were enrolled for hydroxyurea treatment at a low and fixed dose of 10 mg/kg/day. One hundred four patients (Group-I: 37 children and Group-II: 67 adults) with ≥2 years of hydroxyurea treatment were studied. Results The rate of vaso-occlusive crises, requirement of blood transfusion and rate of hospitalization reduced from 3 to 0.5, 1 to 0 and 1 to 0 in Group-I and 3 to 0, 1 to 0 and 0.5 to 0 in Group-II respectively after HU therapy (P < 0.0001). %HbF level, hemoglobin, MCV and MCH increased significantly, whereas HbS, WBC, platelet count, serum-bilirubin and LDH levels decreased significantly after HU therapy. It has been observed that along with fairly subtle hematological changes following HU therapy, there was a substantial clinical improvement occurred in these patients. Transient myelotoxicity was observed in 4.8%. There was minimal gonadal toxicity without affecting reproductive function. Conclusion In view of easy affordability, better acceptability, minimal toxicity, the need of infrequent monitoring and its potential effectiveness, low and fixed dose of hydroxyurea is suitable for treatment of patients with HbSβ+-thalassemia in resource poor setting.Pediatric Blood & Cancer 12/2014; · 2.56 Impact Factor
Candidate Sequence Variants and Fetal Hemoglobin in
Children with Sickle Cell Disease Treated with
Nancy S. Green1*, Katherine L. Ender1, Farzana Pashankar2, Catherine Driscoll3, Patricia J. Giardina4,
Craig A. Mullen5, Lorraine N. Clark6, Deepa Manwani3, Jennifer Crotty1, Sergey Kisselev6,
Kathleen A. Neville7, Carolyn Hoppe8, Sandra Barral9
1Department of Pediatrics, Columbia University, New York, New York, United States of America, 2Department of Pediatrics, Yale University, New Haven, Connecticut,
United States of America, 3Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4Department of Pediatrics, Weill
Cornell University Medical School, New York, New York, United States of America, 5Department of Pediatrics, University of Rochester, Rochester, New York, United States
of America, 6Department of Pathology and Cell Biology, Columbia University, New York, New York, United States of America, 7Department of Pediatrics, Children’s Mercy
Hospitals and Clinics, Kansas City, Missouri, United States of America, 8Department of Hematology-Oncology, Children’s Hospital and Research Center Oakland, Oakland,
California, United States of America, 9G. H. Sergievsky Center, Columbia University, New York, New York, United States of America
Background: Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle
cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle
cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely
attributable to induction of fetal hemoglobin.
Methodology/Principal Findings: In a multi-site observational analysis of children with sickle cell disease, candidate single
nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in
children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated
significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively), with an additive
attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels
explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in
induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels.
Conclusions/Significance: These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin
levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger
populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic
induction of fetal hemoglobin in pediatric sickle cell disease.
Citation: Green NS, Ender KL, Pashankar F, Driscoll C, Giardina PJ, et al. (2013) Candidate Sequence Variants and Fetal Hemoglobin in Children with Sickle Cell
Disease Treated with Hydroxyurea. PLoS ONE 8(2): e55709. doi:10.1371/journal.pone.0055709
Editor: Dimas Tadeu Covas, University of Sao Paulo - USP, Brazil
Received June 28, 2012; Accepted December 29, 2012; Published February 7, 2013
Copyright: ? 2013 Green et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the National Institutes of Health (NIH) to Columbia University from the Best Pharmaceutical for Children’s Act
(3UL1RR024156-04S4 to NSG), the St. Giles Foundation (to Gary M. Brittenham) and a gift from Henry Taub (to NSG). Additional support was provided by NIH
Clinical Translational Science Awards (CTSAs) at Columbia University (5UL1RR024156), Albert Einstein College of Medicine (5UL1RR025750), Weill-Cornell School of
Medicine (5UL1RR024996), University of Rochester (5UL1RR024160), Yale (5UL1RR024139) and Oakland Children’s-UCSF (5UL1RR024131). The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
In sickle cell disease, higher fetal hemoglobin (HbF) levels
diminish de-oxygenated sickle globin polymerization in vitro 
and reduce the incidence of disease morbidities in vivo [2,3]. HbF
is a heritable complex trait [4,5]. Only three genetic loci have been
validated as strongly associated with higher HbF in sickle cell
disease: the 59 beta globin locus (HBB) [5,6,7,8,9], although not
the sickle mutation; the BCL11A repressor of HbF [5,6,7,8,10,11];
and the HS1L-MYB intergenic region [5,6,8,12]. Other candidate
regions have not been confirmed [10,13,14].
Hydroxyurea is the only approved pharmacologic therapy for
sickle cell disease. Its clinical and laboratory effect is understood to
result largely from enhanced HbF expression [3,15,16], although
induction occurs to a highly variable extent [3,4,16,17,18].
Children generally have higher baseline HbF levels than adults
[13,18] and a stable  and overall more robust HbF response to
hydroxyurea [13,18,19]. Hydroxyurea-induced HbF is also
a heritable trait  that generally correlates with baseline levels
[18,19,20]. To date, only limited reports have examined relation-
ships between hydroxyurea-induced HbF and specific genetic
polymorphisms in adults with sickle cell disease [13,21] and did
not explore the recently identified major loci of interest. Genetic
PLOS ONE | www.plosone.org1 February 2013 | Volume 8 | Issue 2 | e55709
determinants for this clinically relevant marker of drug response
have not been confirmed in children [17,19]. Such insight would
be useful for elucidating mechanisms of hydroxyurea induction
and for predicting individual response.
Our multi-site observational study examined associations
between baseline and hydroxyurea-induced HbF in children with
sickle cell disease and candidate single nucleotide polymorphisms
(SNP) in several genes associated with adult sickle cell HbF levels.
Our results indicate: 1) A 33% contribution of baseline to induced
levels; 2) Confirmation of single marker associations between the
HBB and epsilon globin (HBE) and BCL11A loci and baseline HbF
in children; 2) Association between HBE and hydroxyurea-
induced HbF; 3) Additive effects of these SNPs on baseline and
induced HbF in children.
Studies were performed under the policies of and with specific
approval from the Institutional Review Board (IRB) at Columbia
University and the corresponding body at each of the other
participating institutions: Yale University IRB, Albert Einstein
College of Medicine IRB, Weill Cornell University Medical
School IRB, University of Rochester Research Subjects Review
Board, Children’s Mercy Hospitals and Clinics IRB, Children’s
Hospital & Research Center Oakland IRB. Written informed
consent from parents of participating children and informed assent
from children were obtained according to each institution’s IRB
Observational analysis of children attending sickle cell clinic was
performed at five sites (see author affiliations), including pro-
spective observation during 2010. The Oakland site provided
archived data and samples. Clinical inclusion and exclusion
criteria conformed to those previously described [18,22]. Inclusion
criteria are: HbSS or HbS-B0thalassemia, ages 5–21 years.
Exclusions criteria are: pregnancy, current or recent painful crisis,
fever or other acute illness within three weeks prior to evaluation,
transfusion within the prior 100 days or active transfusion therapy,
abnormal elevated serum creatinine or liver transaminases. We
excluded siblings to ensure genetic independence. Laboratory data
at steady state represented the most recent values obtained during
routine care or an average of three values from well visits over the
preceding 6–12 months, if available. Percent HbF (%HbF) was
determined by routine HPLC and was used as a quantitative trait
at steady state for baseline and for drug-induced levels.
Duration of hydroxyurea treatment was for at least six months
and was initiated for comparable clinical indications across sites
(nearly all for repetitive painful crises and/or acute chest episodes).
Stable hydroxyurea dosing was three months at or near maximal
dose by ANC criteria, excluding data from subjects on less than
20 mg/kg/day, even if for dose-limiting toxicity. Across the six
sites, drug dose averages ranged from 23.8–29 mg/kg/day and
did not statistically differ between sites (F=1.554, p=0.210).
Adherence to hydroxyurea was defined as parent report of at least
80% of prescribed doses. Laboratory data at steady state
represented recent values during routine care or averaged three
values from well visits over the preceding 6–12 months, if
available. Percentage of HbF (HbF%) was used as a quantitative
trait. Baseline HbF had not been recorded prior to treatment for
nine of the 47 subjects on hydroxyurea, thus is not available.
Hydroxyurea-associated increased mean red cell volume and
decreased white blood cell count were used to confirm data
quality. For children on hydroxyurea therapy, where available,
clinical response to hydroxyurea was assessed by retrospective
chart review of the number of sickle-related hospitalizations over
the two-year periods preceding and while on hydroxyurea
Description of Procedures
Twenty SNPs previously reported as associated with HbF% in
sickle cell disease [6,7,8,9,11,14,17,19] were genotyped (Table 1):
nine in the BCL11A locus; two in HBS1L-MYB intergenic region
on chromosome 6q23; three in the globin locus on chromosome
11: two 59 sites in HBB (including the previously identified XmnI
site  and one in HBE ; one in OR51 that is upstream of
HBB ; one in the glucagon-like peptide-2 receptor, GLP2R,
found by genome-wide analysis ; two SNPs from the
hydroxyurea-induced SAR1A locus ; one SNP each in ARG1
and ARG2  (SNP sequences available by request).
SNP marker genotyping of minor allele frequencies ranging
from 0.07 to 0.45 was performed using Sequenom MassArray
iPLEX platform with matrix-assisted laser desorption/ionization
time of flight mass spectrometry (Sequenom, San Diego, CA).
PCR assays and mass extension reactions were designed using
mass array assay design software (Sequenom). PCR assays used
Applied Biosystems Geneamp PCR thermocyclers (Foster City,
CA) and analyzed by mass array compact mass spectrometer
(Bruker Daltonik, Billerica, MA) and Spectro TYPER software
(Sequenom). (SNP sequences, PCR and analytic conditions are
available upon request.) Genotyping was performed in duplicate
with separate assays, with genotype frequency distribution at each
SNP tested for deviations from Hardy-Weinberg equilibrium.
Accurate genotyping of SAR1A SNP rs4282891 required sequenc-
ing. The sickle genotype, rs334, was assayed to confirm the
diagnosis for each sample.
Relationships between each of the three quantitative HbF
values were assessed by Pearson correlation analysis. Genetic
associations were assessed for candidate SNPs with %HbF at
baseline, on hydroxyurea treatment (‘‘maximum HbF’’), and the
hydroxyurea-induced increment over baseline (‘‘delta HbF’’)
(Table 1). For each HbF value, quantitative associations with
a dose effect model of the minor allele were tested using a linear
regression analysis, adjusted for sex and age. Baseline HbF levels
were log10 transformed to fit a normal distribution; maximum and
delta HbF were normally distributed. Significance is reported for
nominal p-values of 0.05 or less, and with Bonferroni adjustment
of p-values for multiple testing (adjusted p-value of 361023for the
19 SNPs tested – see below). Given the fixed sample size, power
was estimated based on the smallest detectable differences in the
average levels of baseline and hydroxyurea-induced fetal hemo-
globin HbF. Power was computed using QUANTO software
(http://hydra.usc.edu/gxe/) assuming an additive allele effects
model, an alpha threshold of 0.05, a range of different values for
effect sizes (b) and SNP allele frequency (Figure S2) .
Limited number of subjects of each genotype precluded analysis
of potential dose effect of homozygotes. Trend analysis was
performed by analysis of variance tested for individual and
additive effects for each allele. Percentage of variance attributable
to SNPs in BCL11A, HBB and HBE was estimated using linear
regression analysis comparing models consisting of age, sex,
individual SNPs and multiple SNPs with a reduced model
consisting of sex and age (SPSS Inc., Chicago, IL). To evaluate
whether BCL11A haplotypes was in closer linkage disequilibrium
with causal variants than single SNPs, haplotype association
Sequence Variants with Fetal Hb in Sickle Disease
PLOS ONE | www.plosone.org2February 2013 | Volume 8 | Issue 2 | e55709
analysis was performed with PLINK software  using a haplo-
type block of four BCL11A SNPs in strong linkage disequilibrium
117 children from the six sites met study criteria (Data from
each site are shown in Table S1). The mean age and standard
deviation was 12.5 (SD=4.9), with 52% male. Six subjects (5.1%)
had HbS-B0thalassemia (Table S1A). A subset of children (N=47)
was on hydroxyurea, 38 of whom had recorded baseline and
maximum HbF values. Hydroxyurea dosing ranged from 20–
30.7 mg/kg/day, averaging 25.3 (SD=3.0) mg/kg/day. During
the two years prior to hydroxyurea therapy, children (N=22 with
available data) had 0–13 hospital admissions (mean=3.7,
SD=2.8); only one child had no hospitalizations. During a two
year period while on hydroxyurea therapy, these same children
had 0–6 hospitalizations (mean=1.4,SD=1.5). While on hy-
droxyurea, nearly all had fewer hospitalizations over a two year
period (p=0.001), and 32% had none.
Mean HbF% values were: baseline HbF 8.0 (SD=4.9,
N=108), maximum HbF 18.2 (SD=7.1, N=47), and delta
HbF 11.6 (SD=6.3, N=38). Among those subjects with complete
data for baseline and hydroxyurea-induced HbF (N=38),
distributions of baseline and induced HbF were comparable to
those previously reported for children on hydroxyurea (Figure 1,
Figure S1)  thereby validating our observational data. No
significant effect of gender was detected for any of the three
quantitative traits; age appeared to have some influence on
baseline HbF. Mean baseline HbF at each site ranged from 6.8
(SD=5.0) to 9.8 (SD=5.6), with no significant difference in mean
values (F=0.808, p=0.523), excluding the average value of 12.9%
from Rochester (N=8). At the three sites with at least four subjects
on hydroxyurea (Columbia, Yale and Oakland), no differences
were found in induced maximum or delta HbF (F=0.668,
p=0.418 and F=0.404, p=0.530, respectively).
Pearson correlation for the 38 children with complete baseline
and hydroxyurea-induced HbF demonstrated high levels of
relatedness for: baseline and induced maximum HbF levels
(r2=0.59, p,0.001), similar to previously reported ; maxi-
mum and delta HbF (r2=0.86, p,0.001); but not between
baseline and delta HbF (r2=0.08, p=0.632). Those in the lowest
quartile for baseline levels were highly likely to remain in the
lowest quartile (Q1) for induced HbF (r2=0.565, p,0.001)
(Figure 1). Only two children (5.3%) were induced from the lowest
to highest quartiles (Q3+Q4), and did not appear to differ by age,
sex, sickle type, hydroxyurea dose, genotype, or other discernible
variable, although the small number precluded statistical analysis.
By linear regression analysis adjusted for age and sex, overall
percentage of variance in hydroxyurea-induced HbF attributable
to baseline levels was 33%.
Call rates uniformly exceeded 97% for each SNP. One SAR1A
gene SNP, rs428289,  was monomorphic in subjects treated
with hydroxyurea, precluding analysis of variants. Genotyping of
the other 19 SNPs revealed minor allele frequencies that were
comparable across the six sites (Table S1B) and to allele
frequencies among sickle cell populations in the U.S and elsewhere
Table 1. 19 SNPs associations with baseline, maximum and delta HbF%.
2 BCL11Ars7581162T 0.080.70 0.9080.40 1.550.796
21.43 1.72 0.412HapMap
2 BCL11Ars10189857G0.100.77 0.8980.271.960.891 3.57 2.120.1016
2 BCL11Ars1427407T2.55 0.69*461024
2.14 1.640.2000.191.64 0.909 19
20.20 0.780.8030.87 1.96 0.6594.67 2.130.0356
2 BCL11Ars11886868C2.58 0.64*161024
3.371.380.0191.25 1.42 0.3867,19
2 BCL11A rs4671393A 2.880.68 *561025
3.38 1.590.039 1.20 1.600.459 7,19
2 BCL11A rs7557939G 2.510.64*261024
3.09 1.39 0.0311.101.420.4467,19
2 BCL11A rs10184550G2.24 0.660.0012.44 1.36 0.080
20.561.45 0.701 11
6HBS1L-MYBrs28384513C 0.24 0.900.792
6 HBS1L-MYB rs4895441G
10SAR1A rs2310991A0.050.62 0.942
11 HBBrs10128556T 2.631.34 0.0575.252.860.0742.532.620.3426
11HBB rs7482144A3.88 0.99*261024
11 OR51B6rs5024042A1.700.780.0313.041.93 0.1221.681.850.371 13
Bold indicates SNPs reaching nominally significance (p#0.05).
*Indicates significant SNP after Bonferroni correction for multiple testing.
Sequence Variants with Fetal Hb in Sickle Disease
PLOS ONE | www.plosone.org3 February 2013 | Volume 8 | Issue 2 | e55709
, confirming validity of our pooling strategy. Log10 trans-
formation of baseline HbF had no effect on SNP associations; non-
transformed results are shown for comparison with induced values
Given the fixed sample size and SNP allele
frequencies, the study had 90% power to detect differences in
average levels of baseline HbF% between 2.0 and 2.5 (Figure
S2A). Single marker SNP analysis revealed several significant
associations to baseline HbF (Table 1). One of two SNPs tested
within the HBB locus, rs7482144 , and the HBE SNP  were
significantly associated with baseline HbF after Bonferroni
correction. Six BCL11A SNPs were significantly associated with
baseline HbF, of which five withstood correction for multiple
comparisons, including the intronic variant, rs4671393 [6,7]. To
test for closer linkage disequilibrium with causal variants within
the BCL11A haplotype block than single SNPs , analysis
revealed several common haplotypes (frequency=0.05), with
statistical association and effect size comparable to those seen
with single marker analysis (data not shown). Power was
insufficient to detect effects sizes of ,3, such as for markers in
the HBS1L-MYB intergenic region (Table 1).
Trend analysis using the SNP allele associated with higher
baseline HbF values (‘‘favorable allele’’) in one or both of the
BCL11A (rs4671393) and HBE (rs7130110) loci demonstrated
a statistically significant additive effect of each SNP and a two-fold
difference for both (ptrend,0.004) (Table 2). The HBB marker
(rs7482144) had effects identical to those of HBE (not shown). By
linear regression analysis of individual markers accounting for age
and sex, the percentage of baseline HbF variance attributable each
to the BCL11A or HBE SNP was 13%, and 10% for HBB (Table
S2A). In the multiple marker model, the BCL11A SNP combined
with either globin marker had an additive effect to 21–23%.
Contributions from HBE and HBB appeared to be redundant.
These two markers are in strong linkage disequilibrium within the
globin locus (r2=0.86, p,0.001), with different prevalence of the
minor allele: 18% for HBE and 12% for HBB.
to detect SNPs with effect sizes larger than 2.5 with allele
frequencies of 0.25 or higher; estimated power decreased with
lower SNP allele frequencies (Figure S2B). Among all single
markers tested, only the HBE SNP  was significantly associated
with maximum HbF after correction for multiple testing, and
remained nominally associated even after adjusting for baseline
The study had $80% power
Figure 1. Baseline and hydroxyurea-induced maximum HbF for each subject (comparable to References 18, 19, 26).
Sequence Variants with Fetal Hb in Sickle Disease
PLOS ONE | www.plosone.org4 February 2013 | Volume 8 | Issue 2 | e55709
HbF (p=0.001) (Table 1). This same SNP was also unique in
being associated with the hydroxyurea-induced increment, delta
HbF (p=0.004). Nominal associations were also found between
maximum HbF and four of the same six BCL11A SNPs associated
with baseline HbF (Table 1). However, no association of BCL11A
single marker with maximum HbF withstood adjustment for
baseline HbF (Table 1). Haplotype analysis of BCL11A indicated
nominal association with hydroxyurea-induced HbF, remaining at
borderline significant even after adjustment for baseline HbF (data
By trend analysis, subjects with a favorable allele in at least one
of the BCL11A or HBE loci had higher average values, 20.8
(SD=7.5, N=23), compared to 15.5 (SD=5.6, N=24) for those
without either allele (ptrend=0.009) (Table 2). As with single SNP
analysis, statistical significance of this trend analysis did not
withstand adjustment for baseline fetal hemoglobin (p=0.0.086).
As seen for baseline HbF, the HBB marker (rs7482144) had effects
identical to those of HBE (ptrend=0.018, not shown). By linear
regression analysis of the three markers, alone or in combination,
only the HBE SNP independently and significantly added to the
phenotypic variance contributed by the baseline levels. In
combination, baseline level (33%) and the HBE (13%, p,0.001)
contributed an estimated 46% of the variance (Table S2B).
The GLP2R allele, not associated with baseline HbF, was
nominally associated with maximum HbF; the SAR1A SNP,
rs2310991,  was not.
Children with sickle cell disease generally have higher baseline
HbF levels than adults and more pronounced HbF response to
hydroxyurea [5,6,7,8,9,11,13,16,18,19,20,26]. Findings from this
multi-site, observational analysis corroborate in children the
correlation between baseline and hydroxyurea-induced HbF levels
[18,19], and demonstrate that one third of the variance in induced
HbF is attributable to baseline levels. In contrast to the induced
HbF, the treatment-associated increment appears to be a less
relevant marker, as baseline and delta levels did not correlate and
those with high baseline more likely have highly inducible HbF%.
For baseline HbF, we confirm in children the associations
between single candidate markers in the BCL11A and HBB or
HBE loci . Comparable marker contributions to baseline HbF
were reported for BCL11A and HBB in adult sickle cell disease
[6,7]. In our analysis, statistical effects on HbF from BCL11A and
either globin marker were additive, and were associated with two-
fold higher levels for patients with favorable alleles in both loci. An
apparent redundant influence exists between HBB and HBE
markers, suggesting a statistical effect stemming from tight linkage
and/or overlapping biologic effects.
For hydroxyurea-induced HbF, the HBE marker was the most
robustly associated with drug-induced HbF levels, an effect that
was independent of and additive to baseline levels. Despite
a sample size limiting statistical power for individual SNPs, trend
analysis with BCL11A and HBE or HBB markers suggests
substantially higher induced levels associated with one or both
favorable alleles. In combination with baseline levels, the HBE
accounted for almost half of the variance of induced levels. Taken
together, these data support a model of overlapping genetic
regulation of HbF in pediatric SCD for both steady states .
Roles of other SNP markers are less clear. Nominal association
with GLP2R for hydroxyurea-induced HbF, but not baseline, will
need confirmation and biologic rationale. No associations were
detected with SNPs in other candidate loci such as HBS1L-MYB,
presumably from insufficient power and its lesser impact on
baseline HbF in sickle cell disease . Another hematologic
parameter, alpha-thalassemia status, is not independently associ-
ated with hydroxyurea-induced HbF in children . Effects of
the drug’s pharmaco-kinetics on HbF response are unclear .
Limitations: Study limitations include sample size and observa-
tional assessment, where therapeutic approaches to hydroxyurea
and drug adherence may vary among sites despite inclusion
criteria. Nonetheless, hydroxyurea dose and distribution of drug-
induced HbF were comparable to those from prospective pediatric
hydroxyurea studies . Moreover, a threshold hydroxyurea
dose of 20 mg/kg/day was employed to reduce HbF variability
from low drug dosing.
Clinically, HbF level is arguably the strongest predictor of
disease severity [2,3]. Baseline HbF in children remains the best
forecaster of hydroxyurea-induced HbF but accounts for only
a portion of drug response . Despite a modest sample size,
clinically relevant differences in HbF [2,3] in our study population
appeared to be associated with specific alleles in three genetic loci.
These findings suggest that induced levels in pediatric disease
share some of the major regulatory loci associated with baseline
levels. Our HBE marker data also suggest that induced levels may
reflect effects that are additive to those of baseline. In summary,
our findings suggest that baseline and hydroxyurea-induced HbF
likely are influenced by these genetic loci, while distinct genetic,
pharmacologic, and clinical determinants also exist [18,19].
Genetic studies examining larger pediatric populations on
hydroxyurea and unusual responders are needed to assess the
specific sequence variants in these and other genetic loci
responsible for HbF response.
Histograms for Baseline, Maximum and
quencies. S2A. Baseline HbF. S2B. Maximum HbF
Power analyses based on minor allele fre-
Table 2. Incremental effect of allelotypes in BCL11A and HBE
on Baseline and Maximum HbF%.
rs4671393rs7130110HbF% (SD)HbF% (SD)
297.9 (5.3) 23#
45 6.4 (3.7)*2415.5 (5.6)
Baseline HbF%: Trend analysis across allelotypes: F(df,3)=8.48, ptrend=0.004.
Maximum HbF%: Trend analysis across 0 or $1 A alleles: F(df,2)=7.43,
The + denotes minor allele (A) associated with higher HbF%; the - denotes the
other allele (G),
with the BCL11A A or HBE allele in 1 or 2 copies.
**Not significant when adjusted for baseline HbF.
#Includes subjects with either or both minor A alleles.
Sequence Variants with Fetal Hb in Sickle Disease
PLOS ONE | www.plosone.org5 February 2013 | Volume 8 | Issue 2 | e55709
Clinical data by study site. S1B. Minor allele frequencies by study
Clinical and Genetic Data by Study Site. S1A.
model consisting of age, sex, individual and multiple
SNPs. S2A. Baseline HbF%. S2B. Maximum HbF%
HbF linear regression analysis to compare full
We acknowledge the contributions of Maureen Licursi, MS, CPNP; Mara
Burney, RN, MPH; Columbia’s Human Genetics Resources Core; and the
participating families and coordinators at each site.
Conceived and designed the experiments: NSG KLE SB. Performed the
experiments: NSG KLE FP CD PJG CAM LNC DM JC SK KAN CH
SB. Analyzed the data: NSG KLE LC SK SB. Contributed reagents/
materials/analysis tools: NSG KLE FP CD PJG CAM LNC DM JC SK
KAN CH SB. Wrote the paper: NSG KLE CAM LNC SB.
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Sequence Variants with Fetal Hb in Sickle Disease
PLOS ONE | www.plosone.org6 February 2013 | Volume 8 | Issue 2 | e55709