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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,
1
*Roberto H. Nussenzveig,
2
Gerald Rothstein,
3
Andrew Wilson,
2
Andrew Artz,
4
and
Josef T. Prchal
1,2,5
1
Division of Hematology, School of Medicine, University of Utah, Salt Lake City;
2
ARUP Laboratories, Salt Lake City, UT;
3
Division of Geriatrics, School of Medicine,
University of Utah, Salt Lake City;
4
Section of Hematology-Oncology, University of Chicago, IL; and
5
V eterans Affairs Medical Center, Salt Lake City, UT
Clonality assays, based on X-chromosome
inactivation, discriminate active from inac-
tive alleles. Skewing of X-chromosome al-
lelic usage, based on preferential methyl-
ation of one of the HUMARA alleles, was
reported as evidence of clonal hemato-
poiesis in approximately 30% of elderly
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
thus differentiate clonal malignant and premalignant processes
from reactive polyclonal processes. Detection of clonal cells may
be based on direct tracking of cell lineage–specific sequences or
disease-specific somatic mutations identifying the clonal popula-
tion. Examples include immunoglobin gene rearrangement in
B cells or the 9qϩ;22qϪ translocation in chronic myelogenous
leukemia. Alternatively, clonal populations can be detected through
indirect measures such as the expression of surrogate genes as in
the case of X-chromosome inactivation.
Indirect methods rely on the principle of X-chromosome
inactivation, which is unique in mammals. Most genes in diploid
organisms are expressed from both alleles. However, a subset may
be transcribed preferentially from a single allele. Sex chromosome
gene dosage equivalence between XY males and XX women was
the first described example of an epigenetic process and monoal-
lelic expression.
1
X-chromosome inactivation is a process by which
one of the 2 X chromosomes (or more accurately, most of the genes
on that X chromosome) in each cell is inactivated during early
female embryonic development. The subsequent progeny of each
cell maintains the same inactivated X-chromosome pattern result-
ing in a normal female that is a mosaic of cells; all the coexisting
cells will have either a paternally or maternally derived active
X chromosome. Consequently, establishing clonality requires iden-
tification of X-chromosome polymorphisms. Detection of human
polymorphic X-chromosome genes, subjected to inactivation, was
first described by Beutler et al,
1
and was based on electrophoretic
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
X chromosome will be noted in some women. Skewed X-
inactivation patterns may occur as a result of a primary stochastic
process, or because of secondary cell selection in women heterozy-
gous for certain X-linked genetic diseases.
13,14
Examples of
imbalanced gene expression include random monoallelic (occur-
ring on either autosome or sex chromosome, first foreseen in
1963,
15
and recently found to be more widespread than previously
thought
16
) and imprinting reviewed in Nussenzveig and Prchal.
17
In
mice, genetically determined imbalance of X-chromosome gene
expression based on differences of the X-chromosome inactivation
Submitted March 5, 2008; accepted July 4, 2008. Prepublished online as Blood
First Edition paper, July 18, 2008; DOI 10.1182/blood-2008-03-143925.
*S.I.S., N.A., and R.H.N. contributed equally to this work and should be
considered first authors.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2008 by The American Society of Hematology
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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
The probability that 7 of
8 progenitors of pluripotent hematopoietic stem cells would inactivate
the same X chromosome during embryonic development, resulting in a
skewed allelic ratio of 7:1 and pseudoclonality, is .0078.
10
Based on this
observation and those of others,
11,12
extreme skewing of X-chromosome
allelic usage (allele frequency greater than 80%) is regarded as indicator
of clonality. Although we previously reported preferential allelic usage
of one of the X chromosomes (selection) in women heterozygous
for certain X-linked diseases,
13,14
we have not observed clonal
X-chromosome allelic expression in studies involving more than
200 healthy women, indicating this is a rare phenomenon in the general
population.
10
However, we did not study women older than
65 years.
20-23
To address this issue, we developed a novel quantitative
reverse-transcription allele-specific suppressive PCR (qRT-ASS-PCR),
which is based on a unique primer design,
24
to reinvestigate clonality in
elderly women. We report here the absence of clonal or oligoclonal
hematopoiesis in a group of 37 informative healthy elderly women
between the ages of 65 and 92 years.
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-
tion, PCR amplification with primers flanking both the 5Ј
restriction digestion site and the 3Ј end of the CAG
tandem repeat sequence is performed (Step 4b). Hence,
only intact, methylated, inactive X-chromosome DNA is
amplified. Allele-specific PCR products can be distin-
guished from each other based on the number of
tandem CAG repeats using agarose gel electrophore-
sis (Step 5b).
CLONALITY STUDIES IN ELDERLY WOMEN 3187BLOOD, 15 OCTOBER 2008
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and primary myelofibrosis; these served as positive (disease) control for our
novel clonality assay; and (4) women with nonclonal blood disorders
(including secondary thrombocytosis and leukocytosis); these subjects were
used as negative controls for our novel clonality assay. After informed
consent was obtained in accordance with the Declaration of Helsinki, 5 mL
peripheral blood was collected by venipuncture. Granulocyte, platelet, and
mononuclear cell fractions were isolated by Histopaque (1.077 g/mL)
density gradient based on published protocols.
9
This study received
institutional review board approval from the University of Utah.
Genomic DNA extraction and genotyping of single nucleotide
exonic polymorphisms
Genomic DNA (gDNA) was isolated from granulocytes using the Puregene
DNA purification kit (Gentra, Minneapolis, MN). Genotyping of single
nucleotide exonic polymorphisms from 5 X-chromosome genes (BTK: C/T,
dbSNP: 1135363; FHL1: G/A, dbSNP: 9018; IDS: C/T, dbSNP: 1141608;
G6PD: C/T, dbSNP: 2230037; MPP1: G/T, dbSNP: 1126762)
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
HpaII (undigested samples) was amplified using 2 primers (Table S1)
flanking the STR in the HUMARA gene. One primer was labeled at the 5Ј
end with fluorescein. The PCR products were analyzed and quantified using
an ABI PRISM 3130 Automatic Genetic Analyzer (Applied Biosystems).
Active/inactive X-chromosome allele frequency calculations
Allele frequency of expressed exonic SNPs was calculated as described by
Nussenzveig et al.
24
Briefly, the difference in cycle threshold (⌬C
t
) between
the 2 allele-specific PCR reactions is a measure of the proportion or
frequency of the expressed allele assuming an initial replication efficiency
of 100%. If the amplification efficiencies of the 2 allele-specific reactions
differ slightly, this can be corrected by measuring the ⌬C
t
on a DNA sample
known to be heterozygous for the mutation of interest. The ⌬C
t
in the
heterozygous sample should be 0; any deviation from zero can be
subtracted from all ⌬C
t
measurements to compensate for differing amplifi
-
cation efficiencies and is represented by HC (heterozygote correction
factor). Therefore, HC⌬C
t
represents the heterozygote corrected difference
in cycle threshold between the 2 allele-specific PCR reactions, and is
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
ϩ 1), where E represents the
efficiency of PCR amplification for allele
1
and can be deduced by the slope
of serially diluted sample.
The ratio of the active/inactive X chromosome assessed by HUMARA
assay was determined as described by Bolduc et al.
19
Briefly, the direction
of methylation skewing was determined based on the resulting frequency of
allele A
2
(harbors the greatest number of CAG repeats). In view of
preferential amplification of the smaller allele (A
1
) during PCR, the fraction
of HpaII-digested (A
1
and A
2
) alleles was corrected by the fraction of
undigested (A
1
Ј and A
2
Ј) alleles. These calculations are presented in
Equation 4:
A
2
ϭ 1Ϫ
[
(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))
ϩ
(A
1
/(A
2
ϩA
1
))
(A
1
Ј/(A
2
ЈϩA
1
Ј))
)
]
Statistical analysis1
The data for the analysis were arranged by pairing of the most predominant
allele between markers within a cell lineage (individuals informative for
more than one marker), and by pairing of the most predominant allele of
identical markers and between cell lineages. All statistics were generated
using SAS software, version 9.1 of the SAS system for Windows (SAS
Institute, Cary, NC).
Results
Demographics and genotyping of X-chromosome exonic single
nucleotide polymorphisms in our cohort of healthy women
Genomic DNA was isolated from the peripheral blood granulocytes
of 45 healthy women. Forty of these women were elderly (age in
years: range, 65-92; mean, 81.3; median, 82; coded as GC; Table 1)
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
intention of ascertaining the ⌬Ct values for the 2 possible
3188 SWIERCZEK et al BLOOD, 15 OCTOBER 2008
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genotypes known for each one of the markers. The discrimination
of the X-chromosome allelic usage ratio exceeded ⌬Ct greater than
13 cycles (Table S2) for all X-chromosome polymorphisms used.
Allelic expression ratios of the 5 X-chromosome exonic SNPs
in elderly women
Clonality assay for informative markers was performed using RNA
from freshly isolated platelets and granulocytes. Based on pub-
lished data using the HUMARA assay in elderly women, we
expected to find 12 of 40 elderly women to have skewed allelic
usage. Neither clonal X-chromosome monoallelic expression nor
extreme skewing was noted in any of the study subjects (Table 2).
Moreover, in all of the individuals examined, the X-chromosome
allelic expression ratio of the 5 markers tested was less than 75% of
the predominant allele, well within the limits of variation that were
established in previous studies (Table 2).
10-12
Spearman correlation
coefficients were calculated to assess the linear relationship
between 2 markers in platelets or granulocytes, or for the same
marker between platelet and granulocyte lineages. Statistically
significant correlations were observed, with P value less than .001,
for analysis of 2 different markers and a single hematopoietic
lineage (Figure 2A,B), as well as for analysis of a single marker on
2 different hematopoietic lineages (Figure 2C). Moreover, general
linear models were calculated to test the effects of lineage, marker,
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
and known somatic mutation markers (Table 4). Of these
15 women, 8 patients had polycythemia vera, 4 had essential
thrombocythemia, 1 had primary myelofibrosis, and 2 had primary
myelofibrosis in transformation to acute myeloid leukemia. In
addition, 7 women with nonclonal hematologic disorders were
tested: secondary thrombocytosis (2 patients), secondary erythrocy-
tosis (4 patients), and secondary leukocytosis (1 patient). Using our
novel quantitative transcriptional clonality assay, all patients with a
myeloproliferative disorder were clonal, whereas none of the
patients with secondary conditions were clonal (Table 4).
Discussion
Detection of clonality based on discrimination of the state of DNA
methylation
4
greatly extended the applicability of X-chromosome
Table 1. Age and genotypes of X-chromosome exonic single
nucleotide polymorphisms from our cohort of 45 healthy women
(40 elderly and 5 young)
Volunteer
subjects Age, y
Exonic X-chromosome genetic polymorphisms
tested
MPP1 FHL1 IDS G6PD BTK
GC1 82 G/T G/G C/C T/T C/C
GC2 83 G/G G/A C/C C/C T/T
GC3 89 T/T G/A C/C C/C C/C
GC4 93 G/G G/A T/T C/C C/T
GC5 66 G/G G/A C/T C/C C/C
GC6 67 G/G G/G C/T C/C C/C
GC7 65 G/G G/G C/T C/C C/C
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
GC38 89 G/T G/G C/C C/T C/C
GC39 92 G/G G/G C/C C/T C/C
GC40 92 G/T G/A C/T C/C C/C
YC1 40 G/T G/G C/T T/T T/T
YC2 32 G/G G/A C/T C/C C/T
YC3 33 T/T G/G C/T C/C C/C
YC4 37 G/T G/G C/C C/C C/C
YC5 25 T/T G/A C/T C/C C/C
GC indicates healthy women 65 years or older; and YC, healthy control women
40 years or younger. Italics indicate heterozygous informative subjects.
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Table 2. X-chromosome exonic polymorphism genotypes and their transcribed allelic frequencies in informative healthy women from platelets and granulocytes
Samples
Genotyped X-chromosome polymorphisms
determined to be heterozygous
Allelic frequencies of expressed exonic polymorphisms in
platelets
Allelic frequencies of expressed exonic polymorphisms
granulocytes HUMARA assay
MPP1 FHL1 IDS G6PD BTK MPP1 G/T FHL1 G/A IDS C/T G6PD C/T BTK C/T MPP1 G/T FHL1 G/A IDS C/T G6PD C/T BTK C/T Genotype Allele ratios
GC1 ϩ 54/46 58/42 252/278 9/91
GC2 ϩ 61/39 60/40 - -
GC3 ϩ 63/37 63/37 264/264 -
GC4 ϩϩ56/44 58/42 53/47 55/45 - -
GC5 ϩϩ 53/47 53/47 61/39 60/40 278/289 57/43
GC6 ϩ 40/60 41/59 - -
GC7 ϩ 63/37 60/40 270/281 6/94
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
GC39 ϩ 60/40 58/42 ND
GC40 ϩϩϩ 64/36 30/70 65/35 66/34 25/75 70/30 ND
YC1 ϩϩ 40/60 43/57 43/57 44/56 ND
YC2 ϩϩ ϩ 63/37 35/65 38/62 64/36 34/66 37/63 ND
YC3 ϩ 43/57 45/55 ND
YC4 ϩ 39/61 41/59 ND
YC5 ϩϩ 45/55 51/49 47/53 53/47 ND
Thirty-seven of 40 elderly women (GC) were healthy; 4 of 5 young women (YC) were healthy. The normal range for allelic frequencies was 21% to 80%, determined from more than 200 healthy women.
5
Bold type indicates patients with
skewed allelic methylation by HUMARA assay; blank cells indicate not determined.
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inactivation studies to a broader population. The initial assays for
assessment of clonality using X-chromosome inactivation principle
were hampered by relatively low allelic frequencies of polymor-
phic markers used.
3-6,33
However, this shortcoming was overcome
by the description of the highly polymorphic CAG repeat in the
human androgen-receptor gene (HUMARA assay) that also corre-
lated with X-chromosome inactivation,
34
rendering the majority of
women informative for X-chromosome inactivation assays. Sev-
eral studies involving genetically identical organisms have uncov-
ered profound phenotypical variation. This disparity is at times
compounded by exposure to differing environmental condi-
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
HUMARA technique compared with the analyses of X-chromosome
polymorphic genes used here. Further, interpretation of the methylation
results can be confounded by several factors that can occur during the
Figure 2. Linear regression analysis of the correlation between allelic expres-
sion ratios for 2 markers. PLT indicates platelets; GNC, granulocytes. Comparison
between allelic expression ratios in the same individual, either between markers
within the same lineage (A,B) or between lineages (C). Individuals informative for
more than one marker were used for comparison of the expression ratios in platelets
(A) or granulocytes (B). An identical analysis was performed for comparison of a
single marker between platelet and granulocyte lineages, within the same individual
(C). Excellent correlations were found between compared values obtained in
platelets and granulocytes, with P value less than .001.
Table 3. Statistical analysis and comparison of transcribed exonic
SNPs between elderly and young cohorts of healthy women as a
function of age, marker, and cell lineage
Mean Median SD P
MPP1, n ؍ 7
Age, y 82.6 82.0 6.9 Ͻ.001
PLT allele frequency 64.1 65.0 10.9 Ͼ.05 (NS)
GNC allele frequency 62.7 59.0 9.7 Ͼ.05 (NS)
FHL1, n ؍ 19
Age, y 81.4 82.0 7.6 Ͻ.001
PLT allele frequency 62.8 61.0 6.6 Ͼ.05 (NS)
GNC allele frequency 61.4 61.0 6.0 Ͼ.05 (NS)
IDS, n ؍ 17
Age, y 78.7 77.0 9.1 Ͻ.001
PLT allele frequency 60.4 60.0 6.2 Ͼ.05 (NS)
GNC allele frequency 60.4 60.0 5.6 Ͼ.05 (NS)
G6PD, n ؍ 2
Age, y 92.0 92.0 0.0 -
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
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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
by environmental factors such as drugs, nutrition, and in vitro manipula-
tion of cells during diagnostic testing. Lastly, methylation of inactive
genes is not uniform throughout the inactive X chromosome since many
inactivated genes can be either methylated or hypomethylated.
28,41
Age-dependent skewing in the ratio of allelic methylation at the
X-chromosome–linked HUMARA locus has been reported previ-
ously.
20-23
Likewise, analysis of our cohort of elderly women, using the
methylation-based HUMARA assay, is consistent with age-dependent
skewing at this locus, and preferential methylation of one allele.
Stochastic models of age-dependent skewing at the HUMARA locus
based on contraction of the hematopoietic stem cell pool and clonal
dominance were hypothesized.
22,23
However, recent observations dem
-
onstrate a functional deficit in yet-increased numbers of hematopoietic
stem cells with aging.
42,43
Moreover, reports of accumulating DNA
damage
44
and loss of epigenetic regulation
45
in quiescent aging hemato
-
poietic stem cells may explain this age-dependent skewing. The
molecular mechanism and functional relevance of age-dependent skewed
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
clonality method described here discriminates with high specificity
single nucleotide polymorphisms and allows accurate estimation of the
proportion of active X-chromosome transcripts in a tissue. This permits
not only determination of clonal cells comprising the majority of
circulating cells, but also detection of subclones among circulating
polyclonal cells by comparing allelic usage ratios of platelets and
granulocytes to the circulating long-lived T lymphocytes.
6,10,31
We show that all informative elderly women (Table 2) exhibit
similar ratio of transcribed X-chromosome allelic usage compared
with younger women. In addition, both groups of healthy elderly
and young women differ by a single statistically significant
variable, age. Finally, our data demonstrate that in the absence of
comorbid conditions, most healthy individuals do not exhibit
clonality and oligoclonality of hematopoiesis with aging.
Acknowledgments
The authors acknowledge helpful comments of George Chen and
Alexander Gimelbrant.
This work was supported by 1P01CA108671-O1A2 (National
Cancer Institute, Bethesda, MD) awarded to the Myeloproliferative
Disorders Consortium (PI Ron Hoffman) project no. 1 (PI J.T.P.)
Table 4. Validation of quantitative clonality assay in patients with hematologic disorders
Patient Diagnosis Age, y
Somatic mutations (allele frequency) Allelic frequencies of expressed exonic polymorphism in platelets
JAK2V617F cMPLW515L MPP1 FHL1 IDS BTK G6PD
P1 ET 55 2.2 53.5 88/12
P2 ET 57 24.3 0 1/99
P3 ET 57 19.2 0 98/2 4/96
P4 ET 60 44 0 9/91 96/4
P5 PMF 66 53 0 3/97
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
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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;
A.A. accrued study subjects, participated in study design, and
reviewed the paper; and J.T.P. designed the study, analyzed data,
and wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: J. T. Prchal, University of Utah, School of
Medicine, Hematology Division, SOM 5C210, 30 N 1900 E, Salt
Lake City, UT 84132; e-mail: josef.prchal@hsc.utah.edu.
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HEMATOPOIESIS AND STEM CELLS
Hematopoiesis is not clonal in healthy elderly women
*Sabina I. Swierczek,
1
*Neeraj Agarwal,
1
*Roberto. X-linked diseases,
13,14
we have not observed clonal
X-chromosome allelic expression in studies involving more than
200 healthy women, indicating this is a
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