doi:10.1182/blood-2008-03-143925
Prepublished online July 18, 2008;
2008 112: 3186-3193

Andrew Artz and Josef T. Prchal
Sabina I. Swierczek, Neeraj Agarwal, Roberto H. Nussenzveig, Gerald Rothstein, Andrew Wilson,

Hematopoiesis is not clonal in healthy elderly women
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HEMATOPOIESIS AND STEM CELLS
Hematopoiesis is not clonal in healthy elderly women
*Sabina I. Swierczek,
1
*Neeraj Agarwal,

women. Using a quantitative, transcription-
ally based clonality assay, we reported X-
chromosome–transcribed allelic ratio in
blood cells of healthy women consistent
with random X-inactivation of 8 embryonic
hematopoietic stem cells. Furthermore, we
did not detect clonal hematopoiesis in more
than 200 healthy nonelderly women. In view
of the susceptibility of aging hematopoietic
stem cells to epigenetic dysregulation, we
reinvestigated the issue of clonality in el-
derly women. Forty healthy women (ages
65-92 years; mean, 81.3 years) were tested
by a novel, quantitative polymerase chain
reaction (qPCR) transcriptional clonality as-
say. We did not detect clonal hematopoiesis
in any of the tested subjects. We also tested
DNA from the same granulocyte samples
using the methylation-based HUMARA as-
say, and confirmed previous reports of ap-
proximately 30% extensively skewed or
monoallelic methylation, in agreement
with likely age-related deregulated methyl-
ation of the HUMARA gene locus. We con-
clude that the transcriptionally based X-
chromosome clonality assays are suitable
for evaluation of clonal hematopoiesis in
elderly women. (Blood. 2008;112:3186-3193)
Introduction
Clonality studies can establish the single-cell origin of tumors and

distinction of G6PD isoenzyme products in African women. The
application of G6PD isoenzyme expression for detection of clonal-
ity was first reported in myomas by Linder and Gartler,
2
and then
for malignant tumors by Beutler et al.
3
Vogelstein et al
4
later
proposed detection of clonality by discrimination of the methyl-
ation state of DNA (Figure 1), extending its applicability to most
women regardless of their ethnic origin. Subsequently, other
approaches to identification of the active X chromosome were
developed based on detecting transcribed alleles bearing synony-
mous, or noncoding, single nucleotide polymorphisms (Figure 1),
that is, transcriptional clonality assays.
5,6
The Lyon-Beutler hypothesis of random X-chromosome inacti-
vation provided the basis for assessing hierarchy and clonality of
hematopoiesis.
1,7-9
According to this hypothesis, the ratio of cells
with inactive maternal to paternal X chromosome should follow a
Poisson distribution with a mean around 0.5. The caveat, however,
is that the number of pluripotent stem cells present at the time of
inactivation is small.
10-12
Hence, based on statistical probability, a
skewed ratio between cells with either inactive maternal or paternal

VOLUME 112, NUMBER 8
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locus Xci
18
has been demonstrated; however, a recent study in
humans found this phenomenon to be infrequent since it was not
detected in more than 500 healthy female mother-neonate pairs.
19
Extreme skewing of X-chromosome allelic usage by methylation-
based clonality assay has been reported in approximately 30% of
healthy elderly women,
20-23
and has been attributed to the development
of clonality and oligoclonality as a consequence of hematopoietic stem
cell senescence. Thus, it was recommended that the X-chromosome–
based clonality assays preclude their use in elderly women.
20-23
In
contrast, using a quantitative transcriptionally based clonality assay, we
have previously established that significant skewing of the ratios of
X-chromosome–transcribed alleles is a common occurrence in healthy
women based on our studies demonstrating that 8 progenitors of
pluripotent hematopoietic stem cells are present at the time of random
X-chromosome inactivation in the female embryo.
10
The conclusion
that 8 progenitors of pluripotent hematopoietic stem cells are present at
the time of embryonic random X-chromosome inactivation was corrobo-
rated by others using a different approach.
11,12

Methods
Study subjects
This study included 4 groups of prospectively recruited subjects: (1) healthy
elderly women (Ͼ 65 years of age)—these subjects did not have any active
medical problems and were carefully screened for a history of anemia,
autoimmune diseases, and malignant disorders; (2) younger healthy women
(Ͻ 40 years of age)—age control group for the elderly subjects; (3) women
with clonal myeloproliferative disorders—these subjects had well-
characterized myeloproliferative disorders as per World Health Organiza-
tion criteria
25
and included polycythemia vera, essential thrombocytosis,
Figure 1. Schematic diagram of X-chromosome
clonality determination used here and in HUMARA
assay. X-chromosome inactivation occurs early during
embryogenesis. Hence, women are a mosaic of pater-
nal or maternal active X chromosome (Step 1). Inactive
X chromosome is represented by filled red circles. For
the transcriptional clonality assay, a specific exonic
polymorphism is selected and genotyped (Step 2a).
Allele-specific expression is determined by real-time
PCR using reverse-transcribed mRNA as described in
“Novel transcriptional clonality assay” (Steps 3a and
4a). Resulting amplification curve is used to estimate
the ⌬Ct and corresponding frequencies of each allele
(Step 5a). In contrast, analysis at the HUMARA locus,
shown methylated in the promoter region by filled red
circles (Step 2b), is initiated by restriction digestion
(scissors) of genomic DNA using a methylation-
sensitive endonuclease (Step 3b). After restriction diges-

26
was
determined using TaqMan allele-discrimination assays on an Applied
Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster
City, CA). Briefly, reactions (15 ␮L) consisted of 1 to 20 ng purified gDNA
and 0.75 ␮L TaqMan SNP Genotyping Assay mix (Applied Biosystems);
all other conditions were as described by the manufacturer.
Novel transcriptional clonality assay
Total RNA was isolated from platelets, granulocytes, and T cells using
Tri-Reagent (Molecular Research Center, Cincinnati, OH), and used for
assessment of clonality. Total RNA (50 ng) was reverse transcribed using
SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen,
Carlsbad, CA). Quantitative allele-specific suppressive PCR was performed
on a sequence detection system 7500 platform (Applied Biosystems), using
a modification of previously described method.
24
Typical reactions (15 ␮L)
consisted of 1ϫ TaqMan Universal PCR master mix (Applied Biosystem);
300 nM allele-specific and universal gene–specific primers (Table S1,
available on the Blood website; see the Supplemental Materials link at the
top of the online article); 125 nM FAM-labeled gene-specific MGBNFQ
probe (Applied Biosystems); (Table S1); and first-strand cDNA. Allele-
specific primers were designed using the software program Oligo 6.7
(Molecular Biology Insights, Cascade, CO).
Phenotypic determination of HCI ratios by HUMARA
methylation assay
HUMARA assays were performed as previously described.
19
Briefly, DNA
after digestion with RsaI and HpaII (digested samples) or without RsaI and

computed as described in Equations 1 to 3. Equation 1: ⌬C
t
ϭ C
t-
allele
1
Ϫ C
t-
allele
2
. Equation 2: HC⌬C
t
ϭ⌬C
t
Ϫ (HC C
t-
allele
1
Ϫ HC C
t-
allele
2
). Re
-
sults obtained in Equations 1 and 2 are used to find the frequency of allele
1
in Equation 3: frequency allele
1
ϭ 1/(E
HC⌬Ct

[
(A
2
/(A
2
ϩA
1
))
(A
2
Ј/(A
2
ЈϩA
1
Ј))
(
(A
2
/(A
2
ϩA
1
))
(A
2
Ј/(A
2
ЈϩA
1
Ј))

and 5 were young women (age in years: range, 30-40; mean, 33.4;
median, 33; coded as YC; Table 1). All 45 women were genotyped
to determine zygosity of the 5 X-chromosome exonic polymor-
phisms (Table 1). Forty-two women were informative for 1 or more
tested markers (Table 1). Three women (GC18, GC23, and GC36)
were homozygous (noninformative) for all tested X-chromosome
polymorphic genes (Table 1). The overall heterozygosity of the
polymorphic X-chromosome genes was determined to be 46%,
46%, 19%, 30%, and 5%, for FHL1, IDS, MPP1, BTK, and G6PD,
respectively; these data are in agreement with previously reported
studies using large ethnically diverse populations.
10,26-31
Determination of allele-specific primer specificity and
sensitivity
The difference in ⌬Ct between the 2 allele-specific PCR reactions
is used to estimate allele frequency, assuming initial amplification
efficiency is 100%. The mathematic formulas used to calculate
allele frequencies have been reported elsewhere.
24,32
In these
calculations, the initial 2-fold/cycle is used as a value of 100%
amplification efficiency. Therefore, it can be inferred that the ⌬Ct
between allele-specific reactions reflects fold difference in allele
frequencies. Because initial PCR amplification proceeds at a 2-fold
geometric rate, then the fold difference between allele frequencies
can be estimated by calculating 2
⌬Ct
. To determine the specificity
and quantitative sensitivity of the allele-specific primers, total RNA
from platelets of homozygous women was isolated with the

and age on allele frequency. Models assessing interaction effects were
also computed. Follow-up analysis was performed considering a
categoric divide in age between those younger than 40 years and those
65 years or older. Results of these models comparing our elderly and
young cohorts of healthy women are presented in Table 3.
Comparison between methylation-based HUMARA assay and
our novel quantitative clonality assay in elderly women
Based on reported HUMARA data, approximately 30% of elderly
women were found to have skewed X-chromosome allelic usage (most
prevalent allele frequency greater than 80%). We performed clonality
testing by HUMARA assay in all those elderly subjects whenever
sufficient genomic DNA was available (30 of 40 elderly women). One
of 30 could not be determined due to overlapped PCR stutter peaks;
3 were noninformative (homozygous) based on results from HpaII-
undigested DNA; 9 had skewed (Ͼ 80%) HUMARA-based X-
chromosome allelic usage; and the remaining 17 elderly women had
normal HUMARA-based X-chromosome allelic usage (Table 3). Pres-
ence of skewed allelic methylation ratios in 9 (35%) of 26 informative
elderly women by this assay in our cohort is in agreement with
previously reported literature. In contrast, as already shown, we did not
observe skewed or clonal hematopoiesis in any of these same individu-
als using our novel quantitative transcriptional clonality assay. Formal
statistical analysis, using an exact binomial test, further emphasized the
discrepancy between results obtained using our novel transcriptional
clonality assay and analysis of methylation at the HUMARA locus
(P Ͻ .001; exact 95% CI, 0-0.1).
Validation of quantitative clonality assay in patients with clonal
hematologic disorders
We obtained genomic DNA from peripheral blood granulocytes of
15 women with well-characterized myeloproliferative disorders

GC8 69 T/T G/A C/C T/T C/T
GC9 76 G/G G/A T/T C/C C/C
GC10 82 G/T A/A T/T C/C T/T
GC11 68 G/G G/G C/T C/C C/T
GC12 75 G/T A/A C/T C/C C/C
GC13 73 G/G G/A C/T C/C C/C
GC14 77 G/T G/A C/T C/C T/T
GC15 85 T/T G/A C/T C/C C/C
GC16 78 G/T G/A C/C C/C C/T
GC17 88 T/T A/A C/C C/C C/T
GC18 88 G/G G/G C/C C/C C/C
GC19 91 T/T G/A C/C T/T T/T
GC20 77 G/G A/A C/T C/C C/T
GC21 82 G/G G/G C/T C/C C/C
GC22 90 T/T G/A C/C C/C C/T
GC23 77 G/G G/G T/T C/C C/C
GC24 86 T/T G/A C/C C/C C/C
GC25 85 G/G G/A C/C C/C C/T
GC26 91 G/G G/G T/T C/C C/T
GC27 84 T/T G/G C/T C/C C/C
GC28 77 G/G G/A C/C C/C T/T
GC29 87 T/T A/A C/T C/C C/C
GC30 87 T/T G/A C/C C/C C/T
GC31 76 G/G G/A C/T C/C C/C
GC32 91 G/G G/A C/C C/C C/C
GC33 79 T/T G/G C/T C/C C/C
GC34 92 T/T A/A C/T C/C C/T
GC35 92 T/T G/A C/T C/C C/C
GC36 68 G/G G/G C/C C/C C/C
GC37 74 G/T G/G C/T C/T T/T

GC8 ϩϩ34/66 67/33 36/64 68/32 260/272 68/32
GC9 ϩ 61/39 66/34 272/278 70/30
GC10 ϩ 74/26 73/27 275/289 55/45
GC11 ϩϩ 34/66 32/68 36/64 30/70 267/281 86/14
GC12 ϩϩ 65/35 35/65 59/41 39/61 267/278 92/8
GC13 ϩϩ 42/58 41/59 44/56 48/52 270/275 33/67
GC14 ϩϩϩ 59/41 43/57 52/48 53/47 41/59 52/48 264/270 68/32
GC15 ϩϩ 43/57 54/46 42/58 52/48 - -
GC16 ϩϩ ϩ73/27 30/70 31/69 72/28 35/65 30/70 - -
GC17 ϩ 53/47 54/46 267/272 69/31
GC18 267/278 94/6
GC19 ϩ 27/73 30/70 258/264 9/91
GC20 ϩϩ 70/30 32/68 68/32 34/66 272/289 77/23
GC21 ϩ 48/52 46/54 267/275 91/9
GC22 ϩϩ30/70 31/69 40/60 33/67 272/281 7/93
GC23 289/298 69/31
GC24 ϩ 65/35 66/34 267/281 55/45
GC25 ϩϩ65/35 55/45 57/43 54/46 270/272 36/64
GC26 ϩ 70/30 68/32 270/270 -
GC27 ϩ 30/70 37/63 267/270 47/53
GC28 ϩ 75/25 73/27 272/275 I
GC29 ϩ 61/39 65/35 275/284 60/40
GC30 ϩϩ41/59 57/43 46/54 58/42 275/281 47/53
GC31 ϩϩ 71/29 68/32 68/32 72/28 272/272 -
GC32 ϩ 56/44 63/37 278/284 20/80
GC33 ϩ 60/40 64/36 275/281 40/60
GC34 ϩϩ 56/44 62/38 60/40 64/36 275/284 90/10
GC35 ϩϩ 57/43 54/46 50/50 58/42 267/278 73/27
GC37 ϩϩϩ23/77 36/64 61/39 28/72 38/62 58/42 ND
GC38 ϩϩ48/52 26/74 51/49 30/70 ND

tions.
35,36
Reports of cloned animals with different coat patterns
and behavioral characteristics indicate that the environment plays a
significant role in establishing these traits.
37
Changes in DNA
methylation at CpG islands, which may be associated with
transcriptional silencing, have been linked to an organism’s re-
sponse to environmental factors.
38
For example, studies of DNA
methylation patterns in monozygotic twins found that although
they are epigenetically identical at a young age, as they get older,
differences in the content and distribution of methylcytosine and
associated gene expression diverged.
39
Furthermore, significant
differences in expression phenotypes between twins are observed
at specific chromosomal locations.
40
These locations are character
-
ized by having a low gene density, and usually contain genes that
are involved in cellular response to external signals.
40
Analysis of
1.5 Mb genomic DNA flanking the HUMARA, MPP1, FHL1, IDS,
G6PD, and BTK genes resulted in gene densities of 3, 59, 30, 20, 54, and
36 genes, respectively, a factor that may influence data obtained by

PLT allele frequency 65.0 61.0 7.8 Ͼ.05 (NS)
GNC allele frequency 62.0 58.0 6.9 Ͼ.05 (NS)
BTK, n ؍ 11
Age, y 81.8 82.0 8.8 Ͻ.001
PLT allele frequency 63.3 67.0 6.4 Ͼ.05 (NS)
GNC allele frequency 63.1 66.0 6.5 Ͼ.05 (NS)
Overall GC, n ؍ 56
Age, y* 81.4 82.0 8.2 Ͻ.001
PLT allele frequency 62.5 61.0 7.1 Ͼ.05 (NS)
GNC allele frequency 61.7 61.0 6.5 Ͼ.05 (NS)
Overall YC, n ؍ 9
Age, y* 33.4 33.0 5.7 N/A
PLT allele frequency 59.0 60.0 4.4 N/A
GNC allele frequency 58.4 57.0 4.9 N/A
n represents number of expressed ratios used for calculations that includes
individuals who are informative for more than one marker; NS indicates not
statistically significant.
*Age is not included.
CLONALITY STUDIES IN ELDERLY WOMEN 3191BLOOD, 15 OCTOBER 2008
⅐
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assay, such as incomplete digestion by the methylation-sensitive enzyme.
Quantitation of allelic methylation ratios is further confounded by
difficulty in estimation of the area underneath an allele peak (especially
when alleles are separated by only 3 bp) due to PCR stutter as a
consequence of amplification of small tandem repeats (STRs). More-
over, HUMARA allelic products of different sizes are amplified with
different efficiencies by PCR. In addition, methylation of genes has been
shown to vary over progressive cellular divisions, and can be influenced

methylation at the HUMARA locus remain unanswered, and will have to
be addressed in future studies.
To address the issue of possible clonal evolution of hematopoiesis
with aging, it was necessary to accurately quantify expressed
X-chromosome allelic ratios without introducing a bias resulting in
preferential detection of one of the polymorphic alleles. Previously, we
used a quantitative and reproducible transcriptional clonality assay
based on the ligase detection.
6,10,31
However, this method required use of
large quantities of radiolabeled nucleotide with high specific activity.
Further, ligated products had to be separated on a polyacrylamide gel,
and radioactive bands accurately enumerated by use of a PhosphoIm-
ager (Molecular Dynamics, Sunnyvale, CA). This laborious and hazard-
ous method was subsequently replaced by a simpler, semiquantitative
single-stranded conformational polymorphism method (SSCP).
26
Be
-
cause some X-chromosome genes are only partially or not at all
inactivated,
41
we had to prove that all the genes we studied here are
subject to X-chromosome inactivation and are polymorphic in all major
US ethnic groups, and this was indeed previously documented for the
analyses of X-chromosome exonic polymorphisms used here, that is
FHL1, IDS, MPP1, BTK, and G6PD.
10,26-31
These markers now provide
excellent coverage of all major US ethnic groups. The quantitative

P6 PMF with AML 41 0 0 2/98 2/98
P7 PMF with AML 51 6.2 0 100/0 100/0
P8 PV 8 20 0 96/4 100/0 94/6
P9 PV 49 75.8 0 100/0 95/5
P10 PV 50 15.4 0 100/0 3/97
P11 PV 61 97 0 98/2 100/0 2/98
P12 PV 74 44.5 0 1/99
P13 PV 74 94.3 0 98/2
P14 PV 80 65.6 0 100/0 0/100
P15 PV 90 40.2 - 99/1
P16 Sec erythro 37 0 0 53/47
P17 Sec erythro 42 0 0 42/58 50/50
P18 Sec erythro 47 0 0 58/42
P19 Sec erythro 47 0 0 35/65 52/48
P20 Sec leuko 78 0 0 70/30
P21 Sec thrombo 37 0 0 70/30 60/40
P22 Sec thrombo 46 0 0 30/70
ET indicates essential thrombocythemia PMF, primary myelofibrosis; AML, acute myeloid leukemia; PV, polycythemia vera; Sec, secondary; erythro, erythrocytosis; leuko,
leukocytosis; and thrombo, thrombocytosis. Blank cells indicate not informative and thus not determined.
3192 SWIERCZEK et al BLOOD, 15 OCTOBER 2008
⅐
VOLUME 112, NUMBER 8
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and R01HL50077-14 National Heart, Lung, and Blood Institute,
(PI J.T.P., Molecular Biology of Primary Polycythemia.
Authorship
Contribution: S.I.S., N.A., and R.H.N. designed the study, per-
formed research, analyzed data, and wrote the paper; N.A. and G.R.
accrued study subjects and obtained their consent, and reviewed the
paper; A.W. performed statistical analysis and reviewed the paper;

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