HEMATOPOIESISAND STEM CELLS
Lambert Busque,1-3Yves Paquette,1Sylvie Provost,4Denis-Claude Roy,1-3Ross L. Levine,5Luigina Mollica,1-3and
D. Gary Gilliland6
1Research Centre and2Division of Hematology, Maisonneuve-Rosemont Hospital, Montreal, QC;3University of Montreal, Montreal, QC;4Montreal Heart
Institute, Montreal, QC;5Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY; and6Division of Hematology, Brigham and Women’s
Hospital, Harvard Medical School, Boston, MA
Nonrandom X-chromosome inactivation
(XCI), also known as skewing, has been
documented in the blood cells of a signifi-
cant proportion of normal aging women
by the use of methylation-based assays
at the polymorphic human androgen re-
ceptor locus (HUMARA). Recent data ob-
tained with a new transcription-based XCI
determination method, termed suppres-
sive polymerase chain reaction (PCR),
has shed controversy over the validity of
XCI ratio results obtained with HUMARA.
To resolve this disparity, we analyzed XCI
in polymorphonuclear leukocytes of a
large cohort of women aged 43 to
100 years with the use of HUMARA
(n ? 100), a TaqMan single nucleotide
polymorphism (SNP) assay (n ? 90), and
the suppressive polymerase chain reac-
tion (PCR) assay (n ? 67). The 3 methods
yielded similar skewing incidences (42%,
38%, and 40%, respectively), and highly
concordant XCI ratios. This confirms that
the skewing of XCI ratio seen in blood
cells of aging women is a bona fide and
robust biologic phenomenon. (Blood.
Gene dosage compensation between XX females and XY males
occurs by the random inactivation of one of parental X chromo-
somes in the female embryo.1According to the Lyon hypothesis,1
the fraction of cells with inactivation of each X chromosome
should be equivalent. A deviation from the 1:1 X-chromosome
inactivation (XCI) ratio is referred to as skewing. We have
previously shown that skewing of XCI increases significantly in the
blood cells of female subjects from the neonatal period to old age.2
This observation has subsequently been confirmed by others3-6and
has important implications for understanding the physiology of
etic malignancies. The concordance of skewing documented in
elderly monozygotic versus dizygotic twins7supports a genetic
component to the trait, and work by Abkowitz et al8in a feline
A recent report9argued against the skewing phenomenon in
blood cells of aging women. In this study,9the blood cells of
40 women older than 65 years of age were analyzed by both a
methylation-based assay (ie, human androgen receptor locus
[HUMARA]) and a novel real-time polymerase chain reaction
(PCR) assay, termed suppressive PCR, in which allele-specific
primers are used to identify coding single nucleotide polymor-
phisms (SNPs) in several X-linked genes, including the iduronate-
2-sulphatase gene IDS. The use of HUMARA showed that 30% of
women had skewed XCI in their blood cells, whereas the suppres-
sive PCR assay did not show any significant skewing.9These
observations led the authors to conclude that the skewed XCI
patterns observed with HUMARA were not an accurate reflection
of the proportion of cells bearing a particular X chromosome in the
ation patterns.9Although this explanation is a plausible one for
these data, it is at odds with previously published reports validating
the HUMARA assay against transcription-based assays at the
HUMARA or IDS loci10-12and also with data showing concordance
of skewing at the unlinked HUMARA and phosphoglycerate kinase
To address the disparity suggested by the implementation of the
suppressive PCR assay, we analyzed blood cells of aging women
by using (1) HUMARA, (2) a TaqMan SNP assay at the IDS locus,
and (3) the suppressive PCR assay.9
The study was approved by the Maisonneuve-Rosemont Hospital Ethics
Committee, and informed consent was obtained in accordance with the
Declaration of Helsinki. It included 100 women between 43 and 100 years
of age (mean age, 64 years) who were selected for having a normal
complete blood count and being heterozygous at both the HUMARA and
IDS loci. The methods used to isolate DNA and RNA from polymorpho-
nuclear cells have been previously described.13Herein, skewing was
defined as an XCI ratio ? 3:1. This arbitrary definition has been used in
several other articles.2,13-16
For the HUMARA assay, PCR amplification of the polymorphic CAG
repeat at the HUMARA locus2,17was performed in tandem on undigested
(6-FAM–labeled primer) and on HpaII-digested (HEX-labeled primer)
DNA. Amplification products were migrated on an ABI PRISM 3100
genetic analyzer (Applied Biosystems, Foster City, CA) to determine the
area under the curve (AUC) for each allele. If the alleles are 1 or 2 repeat
lengths different in size, shadow banding from the higher molecular weight
allele (superior allele) can affect the measuredAUC of the lower molecular-
weight allele (inferior allele).18To compensate for this, homozygous
Submitted December 19, 2008; accepted February 2, 2009. Prepublished online as
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samples and samples with more than 5 repeat lengths between the 2 alleles
were used to measure the percentage of shadow banding for each repeat
length, and this percentage was subtracted from the inferior allele. The XCI
ratios determined by the HUMARAassay are reported using the percentage
of cells with the superior allele active (%Sup), and is calculated as follows:
% Sup ? 1 ?[
(A/A ? a)
(A?/A? ? a?)
(A/A ? a)
(A?/A? ? a?)?
(a/A ? a)
(a?/A? ? a?)]
(A and A? represent the AUC of the superior HUMARA allele from the
digested and undigested sample, respectively. The AUC of the inferior
HUMARA allele for the digested and undigested samples are represented by
a and a?, respectively.)
A %Sup of 50% indicates no skewing, a %Sup of 100% indicates that
the superior allele is always active, and a %Sup of 0% indicates the opposite.
Skewing is said to be present if %Sup is equal to or greater than 75% (in cases
where the superior allele is the predominant allele) or equal to or less than 25%
We used 2 transcription-based assays in this study. The first was the
TaqMan SNPassay,11which is based on the detection of a exonic SNPof the
IDS gene (reference ID: rs1141608). For this assay, cDNAwas synthesized
from PMN RNA with the use of random hexamers and TaqMan Reverse
Transcription Reagents (Applied Biosystems), after which the aforemen-
tioned SNP was detected with the use of the TaqMan SNP assay (Applied
Biosystems). This assay included 2 probes capable of distinguishing each
allele of the SNPin real time in the same PCR and was conducted on anABI
Prism 7000 machine (Applied Biosystems). The second transcription-based
assay was the suppressive PCR assay at the IDS locus. For this assay, cDNA
was synthesized from PMN RNA by use of the Omniscript Reverse
Transcriptase kit (QIAGEN, Mississauga, ON). PCRs were performed in
separate tubes for the C and T alleles with the cIDS-R reverse primer and
either the cIDS-LNA-C-F or the cIDS-LNA-T-F forward primer (Integrated
DNA Technologies, Coralville, IA), as described by Swierczek et al.9
Amplified products were detected using the Platinum SYBR green PCR
Supermix (Invitrogen, Mississauga, ON). Real-time PCR was performed on
a Bio-Rad iCycler IQ system (Bio-Rad Laboratories, Mississauga, ON).
PCR parameters were 50°C for 2 minutes, 95°C for 10 minutes, and
50 cycles of 94°C for 10 seconds, 45°C for 20 seconds, and 72°C for
30 seconds. Ct values were determined by use of iCycler software (Bio-Rad
Laboratories). Allele frequencies were calculated from a standard curve
generated from cDNA prepared from cell mixtures (2 men hemizygous for
the different alleles [C vs T]). Subject samples were measured in triplicate,
and mean ?Ct value used to calculate allele frequency. A second standard
curve was obtained from mixtures of PCR fragments obtained from
hemizygous men using cIDS-R and a forward primer located 30 bases
upstream of the SNP (cIDS-F). For both transcription-based assays,
samples in which 75% or more of cells express the same IDS allele are said
to be skewed. This is in line with our definition of skewing (XCI
ratio ? 3:1).
Statistical analysis (Pearson correlation, ?2) was conducted with the use
of NCSS 2004 (NCSS, Kaysville, UT). Agreement and repeatability
coefficients were determined by use of the Bland-Altman method.19
Results and discussion
Repeatability of the 3 methods
The reproducibility of results obtained with each of the 3 assays
was evaluated by the “coefficient of repeatability” described by
Bland and Altman,19which includes 95% of the differences between
2 repeated measurements. The coefficient of repeatability19was 0.0332
(3.3%) for the HUMARA assay, 0.0640 (6.4%) for the TaqMan SNP
assay, and 0.2302 (23.0%) for the suppressive PCR assay. Thus, the
results obtained with the suppressive PCR assay were less reproducible
Incidence of skewing with 3 methods
We evaluated %Sup in the blood cells of 100 women by using
HUMARA. Adequate RNA samples were available for 90 of them
when we performed the TaqMan SNP assay and for 67 when we
performed the suppressive PCR assay. The incidence of skewing
(%Sup ? 75% or ? 25%; percentage of 1 of the 2 IDS alleles
? 75%) was similar with all assays: 42/100 (42%) with HU-
MARA, 34/90 (38%) with the TaqMan SNP assay, and 27/67
(40%) with the suppressive PCR assay. There was no statistically
significant difference when comparing any 2 of these assays, or the
group of 3 (?2; P ? .84). The incidence also was similar in the
subgroup of women older than 65 years (mean age, 71 years): 16 of
40 (40%) with HUMARA, 15 of 36 (41.7%) with TaqMan SNP
assay, and 9 of 22 (40.9%) with the suppressive PCR assay.
Intraindividual concordance of XCI patterns obtained with
XCI patterns obtained with HUMARA were compared with those
obtained with 2 transcription-based assays (Figure 1A,B). We
Figure 1. Correlation of XCI patterns obtained with the use of 3 different methods. (A) HUMARAassay versus TaqMan SNP assay at the IDS locus. For each individual,
the %Sup measured by HUMARAwas compared with the percentage of IDS allele obtained with theTaqMan SNPassay, which was quantitatively closer to the HUMARAvalue.
Data from both assays were available for 90 women. (B) HUMARA assay versus suppressive PCR assay at the IDS locus. The %Sup obtained by HUMARA was compared
with the percentage of IDS allele obtained with the suppressive PCR assay, which was quantitatively closer to the HUMARA value. Data from both assays were available for
67 women. (C) TaqMan SNP assay versus suppressive PCR assay. The percentage of IDS allele obtained with the TaqMan SNP assay was compared with that obtained with
the suppressive PCR assay. Data from both assays were available for 60 women. Those aged 44 to 65 years are represented by ‚, whereas those older than 65 years are
represented by F.
SKEWING IN BLOOD CELLS OFAGING WOMEN3473BLOOD, 9APRIL 2009?VOLUME 113, NUMBER 15
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observed highly concordant results between HUMARA and Taq-
Man SNP: mean of differences was 0.5% (standard deviation
[SD] ? 6.7%), mean of the absolute value of the differences was
5.4%, and correlation was 96%. Similarly, HUMARAand suppres-
sive PCR methods gave concordant results: mean difference was
1.6% (SD ? 12.2%), mean of the absolute value of the difference
was 8.5%, and correlation was 90%. TaqMan SNP and suppressive
PCR methods also were concordant (Figure 1C).
Confirmation that XCI skewing documented by HUMARA
We show that measurements of XCI patterns by HUMARA are
highly concordant with those obtained with 2 transcription-based
methods, including the suppressive PCR assay. Our data should
dissipate concerns about potential age-associated changes in meth-
ylation patterns that could influence HUMARAresults. In so doing,
the data presented herein validate the use of HUMARA for the
study of X-inactivation in elderly females.
Although we observed a poorer repeatability of the suppressive
PCR assay (compared with the other 2 assays), in our hands, the
suppressive PCR assay gave results that correlated well with
previously established XCI determination methods. Therefore, we
find it difficult to explain the discordant data reported by Swierczek
et al.9Unlike Swierczek et al,9we used SYBR green instead of the
generic TaqMan probe (nonallele specific) for the real-time quanti-
fication. It is possible that an interaction between theTaqMan probe
and the suppressive primers played a role in the discordant results.
Moreover, the suppressive approach has no significant advantage
over the TaqMan SNP assays, which use 2 different allele-specific
probes in the same reaction, ensuring identical amplification
conditions for the 2 alleles. The suppressive PCR approach, which
uses allele-specific primers to amplify the 2 different SNPalleles in
separate PCRs,9is per se more subject to amplification biases. In
fact, the standard curve obtained with cIDS-F primer indicated that
with the PCR conditions used, the cIDS-LNA-T-F primer amplified
only 1.2% of the T allele present in the reaction tube, whereas the
cIDS-LNA-C-F amplified 2.9% of the C allele, indicating a strong
bias in amplification efficiency for the 2 alleles. It is also important
to note that the analysis of pure clonal or homozygous patients9is
not sufficient as validation or standardization for this assay.
Support for the acquired skewing phenomenon
Although this study was designed to ascertain the validity of the
HUMARA method for determining XCI patterns in aging women,
it indirectly validates previous studies in which HUMARA assay
documented age-dependent increases in skewing incidence in
females.2-4,6,20Furthermore, if only transcription based analysis
were to be taken into account, the results obtained in this cohort of
aging females and our recently published data obtained in neo-
nates13document that the incidence of skewing triples from birth to
older age. Further studies on age-dependent skewing may help
understand the biology of the aging hematopoiesis, and the
pathobiology of hematopoietic malignancies of the elderly.
L.M. is a Fonds de la Recherche en Sante ´ du Que ´bec (FRSQ,
Montre ´al, QC) scholar.
Contribution: L.B. designed research and wrote the paper; Y.P.
performed research; S.P. analyzed data; D.-C.R. designed research;
R.L.L. reviewed the paper; L.M. designed the study and wrote the
paper; and D.G.G. wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Lambert Busque, MD, FRCPC, De ´partement
d’He ´matologie, Ho ˆpital Maisonneuve-Rosemont, 5415, Boulevard
de l’Assomption, Montre ´al, QC, Canada H1T 2M4; e-mail:
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3474 BUSQUE et alBLOOD, 9APRIL 2009?VOLUME 113, NUMBER 15
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online February 6, 2009
2009 113: 3472-3474
and D. Gary Gilliland
Lambert Busque, Yves Paquette, Sylvie Provost, Denis-Claude Roy, Ross L. Levine, Luigina Mollica
confirmed by independent methodologies
Skewing of X-inactivation ratios in blood cells of aging women is
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