Hematopoiesis is not clonal in healthy elderly women pptx

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

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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 is not clonal in healthy elderly women

*Sabina I Swierczek,1*Neeraj Agarwal,1*Roberto H Nussenzveig,2Gerald Rothstein,3Andrew Wilson,2Andrew Artz,4and Josef T Prchal1,2,5

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 Veterans 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 9q⫹;22q⫺ 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.1X-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 coexistresult-ing

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,1and 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,2and then for malignant tumors by Beutler et al.3 Vogelstein et al4 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.1,7-9According 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.10-12Hence, 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

1963,15and recently found to be more widespread than previously thought16) and imprinting reviewed in Nussenzveig and Prchal.17In 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.

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locus Xci18 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,20-23and 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.10The 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.11,12The 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 0078.10Based on this

observation and those of others,11,12extreme 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.20-23To 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,24to 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 (⬎ 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 (⬍ 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 criteria25and 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 materpater-nal 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 5⬘

restriction digestion site and the 3 ⬘ 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 3187 BLOOD, 15 OCTOBER 2008䡠VOLUME 112, NUMBER 8

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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.24Typical reactions (15␮L)

consisted of 1⫻ 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.19Briefly, 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 5⬘

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.24Briefly, 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 ton a DNA sample

known to be heterozygous for the mutation of interest The⌬C tin the

heterozygous sample should be 0; any deviation from zero can be

subtracted from all⌬C tmeasurements to compensate for differing

amplifi-cation efficiencies and is represented by HC (heterozygote correction

factor) Therefore, HC ⌬C trepresents 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:⌬Ct⫽ Ct-allele1⫺ C

t-allele2 Equation 2: HC ⌬C t ⫽ ⌬C t ⫺ (HC C t- allele 1 ⫺ HC C t- allele 2)

Re-sults obtained in Equations 1 and 2 are used to find the frequency of allele1

in Equation 3: frequency allele ⫽ 1/(EHC⌬Ct⫹ 1), where E represents the

efficiency of PCR amplification for allele1and 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.19Briefly, 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 ⬘ and A 2⬘) alleles These calculations are presented in Equation 4:

A2⫽ 1⫺[ (A2/(A2⫹A1))

(A2⬘/(A2⬘⫹A1⬘))

(A2⬘/(A2⬘⫹A1⬘))⫹

(A1/(A2⫹A1))

(A1⬘/(A2⬘⫹A1⬘))) ]

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

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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).10-12Spearman 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 (⬎ 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⬍ 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 leukocyerythrocy-tosis (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 methylation4greatly 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

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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5 Bold

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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.3-6,33However, 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,34rendering 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.38For 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.40These locations are character-ized by having a low gene density, and usually contain genes that are involved in cellular response to external signals.40Analysis 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 ⴝ 7

PLT allele frequency 64.1 65.0 10.9 ⬎.05 (NS) GNC allele frequency 62.7 59.0 9.7 ⬎.05 (NS)

FHL1, n ⴝ 19

IDS, n ⴝ 17

G6PD, n ⴝ 2

-PLT allele frequency 65.0 61.0 7.8 ⬎.05 (NS) GNC allele frequency 62.0 58.0 6.9 ⬎.05 (NS)

BTK, n ⴝ 11

Overall GC, n ⴝ 56

Overall YC, n ⴝ 9

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.

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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, methylamanipula-tion 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.20-23Likewise, 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.22,23However, recent observations

dem-onstrate a functional deficit in yet-increased numbers of hematopoietic

stem cells with aging.42,43 Moreover, reports of accumulating DNA

damage44and loss of epigenetic regulation45in 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.6,10,31However, 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,41we 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

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

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.

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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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From

Tiêu đề	Hematopoiesis Is Not Clonal In Healthy Elderly Women
Tác giả	Sabina I. Swierczek, Neeraj Agarwal, Roberto H. Nussenzveig, Gerald Rothstein, Andrew Wilson, Andrew Artz, Josef T. Prchal
Trường học	University of Utah
Chuyên ngành	Hematology
Thể loại	Bài báo
Năm xuất bản	2008
Thành phố	Salt Lake City

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Số trang	9
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