A QTL influencing F cell
production maps to a gene
encoding a zinc-finger protein
on chromosome 2p15
Stephan Menzel1, Chad Garner2, Ivo Gut3, Fumihiko Matsuda3,
Masao Yamaguchi3, Simon Heath3, Mario Foglio3,
Diana Zelenika3, Anne Boland3, Helen Rooks1, Steve Best1,
Tim D Spector4, Martin Farrall5, Mark Lathrop3&
Swee Lay Thein1,6
F cells measure the presence of fetal hemoglobin, a heritable
quantitative trait in adults that accounts for substantial
phenotypic diversity of sickle cell disease and b thalassemia.
We applied a genome-wide association mapping strategy to
individuals with contrasting extreme trait values and mapped
a new F cell quantitative trait locus to BCL11A, which
encodes a zinc-finger protein, on chromosome 2p15. The
2p15 BCL11A quantitative trait locus accounts for 15.1%
of the trait variance.
Genome-wide association methodology has recently identified sus-
ceptibility loci for several diseases, but it has a relatively high per-
sample cost and requires large samples to detect modest risk effects.
Strategies to increase power include selecting subjects with increased
genetic load through early onset or identifying familial clustering of
disease. Here, we apply a powerful alternative approach that uses a
comparatively small number of study subjects taken from the extremes
of a quantitative distribution.
In healthy adults, fetal hemoglobin (HbF; also known as a2g2) is
present at residual levels (o0.6% of total hemoglobin) with over
twenty-fold variation. Ten to fifteen percent of adults in the upper tail
of the distribution have HbF levels between 0.8% and 5.0%, a
condition referred to as heterocellular hereditary persistence of fetal
hemoglobin (hHPFH)1. Although these HbF levels are modest in
otherwise healthy individuals, interaction of hHPFH with b thalasse-
mia or sickle cell disease (SCD) can increase HbF output in these
individuals to levels that are clinically beneficial2. The ameliorating
effect of HbF on SCD and b thalassemia has prompted numerous
genetic and pharmacological approaches to reactivation of HbF
synthesis3, but the molecular mechanisms are not fully understood.
Current pharmacological agents, such as hydroxycarbamide and
butyrate analogs, show that it is possible to augment HbF production
therapeutically, but these agents are limited by toxic effects and
variable patient response.
HbF in the normal range (including hHPFH) is most sensitively
measured by the proportion of F cells (that is, the proportion of
erythrocytes containing measurable amounts of HbF1). The majority
of the quantitative variation is highly heritable (h2¼ 0.89)4, but the
genetic etiology is complex, with several contributing quantitative trait
loci (QTLs). To date, major QTLs have been identified with strong and
reproducible statistical support at XmnI-Gg in the b globin locus on
chromosome 11p15 (ref. 5) and in the HBS1L-MYB intergenic region
on chromosome 6q23 (ref. 6).
To map additional QTLs, we selected a panel of 179 unrelated
individuals from the extreme upper and lower tails (above the 95thor
below the 5thpercentile points (that is, 4P95or oP5)) of the F cell
distribution, drawn from a database of 5,184 phenotyped indivi-
duals from the St. Thomas Adult Twin Registry (http://www.
twinsuk.ac.uk7), and genotyped them using the Illumina Sentrix
HumanHap300 BeadChip (Supplementary Methods online). The
study was approved by the local ethics committee of St. Thomas’
and King’s College Hospitals, London (LREC number 00-245), and all
participants gave informed written consent. For the 308,015 markers
retained after quality control, we assessed association using a Fisher
exact w2statistic for the allele counts in the high or low trait categories
along with a linear regression analysis of the continuous trait against
genotype (additive effects), with age and sex included as covariates.
The two analyses gave similar results, and P values from the allele
count test are presented in the text. Tests of non-additivity in the
linear regression led to identical conclusions. Although extreme
discordant sampling designs violate the usual normality assumption
of linear regression, it does not inflate the type 1 error rate8, which we
confirmed by simulations and inspection of the Q-Q plot (Supple-
mentary Fig. 1 online). The genomic control parameter was 1.01,
indicating that there was minimal admixture or cryptic relatedness in
this sample9. Principal components analysis10confirmed this.
We identified major QTLs on chromosomes 2p15 (P ¼ 4.0 ?
10–16), 6q23 (P ¼ 8.8 ? 10–25) and 11p15 (P ¼ 1.7 ? 10–26) (Fig. 1a).
The 6q23 QTL was first localized through linkage analysis in a large
Asian-Indian family with beta thalassemia11, then validated and fine-
mapped in northern Europeans6. The association signal on 11p15
maps to the beta globin cluster, where the functional variant is
thought to be the XmnI-Gg variant at position –158 upstream of the
Gg globin gene5.
Markers within a 126-kb segment on chromosome 2p15 (nucleo-
tides 60456396 to 60582798) identified a third, previously unreported
QTL close to the oncogene BCL11A12. We genotyped an additional
Received 15 March; accepted 2 July; published online 2 September 2007; doi:10.1038/ng2108
1King’s College London School of Medicine, Division of Gene and Cell Based Therapy, King’s Denmark Hill Campus, London SE5 9PJ, UK.2University of California at
Irvine, Epidemiology Division, Department of Medicine, Irvine, California 92697-7550, USA.3Centre National de Ge ´notypage, Institut Ge ´nomique, Commissariat a `
l’Energie Atomique, 91006 Evry, France.4King’s College London School of Medicine, Division of Genetics and Molecular Medicine, St. Thomas’ Hospital, London SE1
7EH, UK.5The Wellcome Trust Centre for Human Genetics, Department of Cardiovascular Medicine, University of Oxford, Headington, Oxford OX3 7BN, UK.6King’s
College Hospital, Department of Haematological Medicine, Denmark Hill, London SE5 9RS, UK. Correspondence should be addressed to S.L.T. (firstname.lastname@example.org).
NATURE GENETICS VOLUME 39 [ NUMBER 10 [ OCTOBER 20071197
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
142 SNPs, 103 of which came from HapMap13and 39 of which were
identified from dbSNP or by resequencing (Supplementary Table 1
online). Analysis of this dense marker set uncovered two clusters of
markers showing highly significant association at P o 10–10(Fig. 1b).
The strongest associations (for example, P o 10–19at rs1427407) were
in a region spanning 14 kb at nucleotides 60561398 to 60575745 in the
second intron of BCL11A. The second association cluster spanned
67 kb at nucleotides 60457454 to 60523981 in the 3¢ region of the
gene, located approximately 8–74 kb downstream of exon 5. Markers
that were significantly associated with the trait generally showed high
LD within each cluster and lower LD between clusters (Supplemen-
tary Fig. 2 online).
To corroborate our findings, we investigated two additional sample
panels (the ‘replication panel’ and the ‘twin panel’, as defined below)
with markers selected to represent the three QTLs (Table 1). For
chromosome 2p15, we examined four markers from the first associa-
tion cluster and two markers from the second association cluster. For
6q23, we chose markers from three linkage disequilibrium groups that
contribute independently6. The XmnI-Gg marker was genotyped
First, we replicated the associations in an independent group of 90
individuals with contrasting trait values (replication panel, n ¼ 90,
oP5or 4P95) (Table 1). Then, we measured the contribution of the
marker to the overall trait variance in an unselected group of 720
twins (‘unselected twins’; 310 dizygotic twin pairs and 100 mono-
zygotic twin pairs) (Table 1). As related individuals were included, we
applied a mixed linear model to test association and estimate residual
heritability in the twin panel. The individual markers were all
significantly associated with the trait (Table 1). A within-family test
of association14, which has less power but controls for possible
population stratification, was significant for markers at the chromo-
some 2 and chromosome 6 QTLs. The trait variance attributed to each
locus in the mixed linear model was 15.1% (95% confidence interval
(c.i.) 12.6%–17.6%) for 2p15, 19.4% (16.6%–22.2%) for 6q22 and
10.2% (8.2%–12.2%) for 11p15. Tests of interactions between QTLs
were nonsignificant, suggesting that they contribute additively.
Together, they explain over 44% of the total trait variance in the
twin panel (that is, half of the overall heritability of 89%).
Table 1 Results for representative markers for the three principal F cell QTLs
Contributions to F cell variation (%)
Allele frequency Association test (P value)N ¼ 720
Low / high F cell GWA and
N ¼ 179
N ¼ 90
N ¼ 269
0.44 / 0.72
0.41 / 0.71
0.19 / 0.59
0.03 / 0.42
0.03 / 0.41
0.03 / 0.41
0.35 / 0.56
0.18 / 0.57
0.38 / 0.10
5,232,7450.330.10 / 0.63 2.0E-304.0E-11 2.4E-3810.2 1.2E-17n.s.10.2
The within-family association test has been included for completeness. It is used principally in presence of population stratification (not found in our sample). It has less power than
the ANOVA test and does not incorporate information on monozygotic twin pairs. n.s., not significant.
aMarkers used to estimate the locus’s contribution to the variance.bMarkers not part of the genome-wide SNP set.cThe within-family association test calculated with the QTDT program.
10 11 12
13 14 151617 18 19 20 21 22 X
60300000 60400000 60500000 60600000
Chromosome 2p15 location (bp)
60700000 60800000 60900000
Figure 1 Association statistics (?log10(P)) for individuals included in the
genome-wide screening panel. (a) Association statistics for 3,225 markers
genome-wide with P o 10–2. (b) Association statistics for 211 markers
across the 2p15 region of association.
1198VOLUME 39 [ NUMBER 10 [ OCTOBER 2007 NATURE GENETICS
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
Haplotype analysis in the twin panel showed incomplete linkage Download full-text
disequilibrium, particularly between markers in the two association
clusters (Supplementary Tables 2 and 3 online). A forward stepwise
regression identified two markers (rs4671393 and rs6732518) from the
first association cluster showing independent statistical effects on the
trait. In particular, the markers from the second cluster did not show
significant association after taking into account rs4671393 and
rs6732518 (Supplementary Table 4 online). This is consistent with
the presence of more than one functional SNP or with the presence of
untyped functional SNPs in incomplete LD with the typed markers
from the first association cluster.
Accumulating experimental data are uncovering the genetic archi-
tecture of human quantitative variation. Resequencing studies of
candidate genes in extreme groups have found diverse sets of rare,
nonsynonymous alleles that collectively explain a modest proportion
of the trait variance for some QTLs, whereas other QTLs are associated
with common alleles—for example, circulating angiotensin 1 convert-
ing enzyme (ACE) activity. Applying GWA to individuals with
contrasting extreme quantitative trait values is a powerful strategy
for mapping common QTLs, as illustrated by our identification of
three principal QTLs that contribute to F cells (and thus HbF).
One of the QTLs that we have identified is a new locus that maps to
the gene encoding the C2H2-type zinc-finger protein BCL11A on
chromosome 2p15, previously implicated in myeloid leukemia and
lymphoma pathogenesis12. We examined multiple tissue cDNA panels
by RT-PCR and found BCL11A to be expressed in a variety of tissues,
including erythroid cells (Supplementary Fig. 3 online). Mouse
studies have shown that Bcl11a is essential for early lineage commit-
ment in the development of T and B cells12. BCL11A has also been
implicated in histone deacetylation and transcriptional repression
in mammalian cells15. We speculate that dysregulated BCL11A
expression may influence F cell production by affecting the kinetics
It is likely that we have identified the principal QTLs that have
frequent alleles affecting F cell production in the general European
population, within the limits of the genome coverage of our
markers. It is possible that additional loci could be uncovered with
a denser map, but most of the remaining heritability is probably
due to multiple small QTLs. The loci uncovered here have a major
influence on the quantitative variation of the trait in healthy adults
and possibly on the ‘erythropoietic stress’ responses underlying
variability in b thalassemia and sickle cell disease severity and on
the capacity of affected individuals to respond to pharmacologic
inducers of HbF.
Note: Supplementary information is available on the Nature Genetics website.
We thank C. Steward for help in preparation of the manuscript. This work was
supported by a grant from the UK Medical Research Council (MRC; G0000111
and ID 51640) to S.L.T. and by the French Ministry of Higher Education and
Research (M.L.). Twins UK is supported by the Wellcome Trust and Framework
V EU (European Union) grant ‘Genome EU Twin’.
S.M. performed research, analyzed data and wrote the paper; C.G. analyzed data;
M.Y. and M. Foglio performed bioinformatics analyses; I.G. and S.H. performed
genome-wide association genotyping; D.Z., A.B., H.R., and S.B. performed
research; T.D.S. contributed material; M. Farrall performed statistical genetic
analysis and wrote the paper; M.L. codirected the research, analyzed data and
wrote the paper; S.L.T. codirected the research and wrote the paper.
Published online at http://www.nature.com/naturegenetics
Reprints and permissions information is available online at http://npg.nature.com/
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