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CORRESPONDENCE
269
TRANSCRIPTIONAL ANALYSIS OF THE ACTIVE X-CHROMOSOME IN NORMAL
AND CLONAL HEMATOPOIESIS
To the Editor:
is conservative. The distribution of this silent polymorphism includes
all races and ethnic groups, suggestingthat it is the result of an ancient
The concept of clonality has been central to the understanding of
tumor development,’ as well as to an understanding of hematopoiesis
and hematopoietic stem cell hierarchy?4 Most clonality assays use
the fact that female cells have only one active X-chromosome. The
other is inactivated in early embryogenesis and remains inactive in
all subsequent somatic progeny. Because the inactivation process is
random, normal female tissue is a mosaic of cells, whereas clonal
tissue is composed of cells bearing the same active X-chromosome.
Peptide polymorphisms encoded by genes on the X-chromosome
have been used to determine if one or two gene products are present
in tissue.’ More recently, Vogelstein et al’ assayed for restriction fragment X-chromosome DNA polymorphisms and detected clonality
by differences in the level of methylation in active and inactive chromosomes. In this approach, treatment of one aliquot of genomic
DNA with a methylation sensitive restriction enzyme leads to the
disappearanceof one allelic restriction fragment in clonal tissue, but
not in normal tissue. The limitation of this technique lies in the
incomplete correlation between different probes for more than one
allele in female heterozygotes? This may be caused by incomplete
methylation of some DNA sequences. To circumvent these limitations, we have developed an assay based on the C T polymorphism
present in exonic nucleotide 131 1 of the X-chromosome gene G-6PD.’ Analysis of mRNA transcriptsin cells from heterozygous females
allows detection of the active X-chromosome.
Single-base mutations may not change the peptide sequence if they
occur in noncoding portions of the gene, such as introns, or if they
result in conservative exon mutations. The common G6PD C T
polymorphism present in exon no. 1 1 at position 131 1 of this gene
mutation!
For the preparation and isolation of cells, the myeloid cells and
cultured fibroblasts were separated as previously described: lymphocytes were prepared by sorting nonadherent mononuclear cells’
and selecting for CD3-/CD 19- antigen for NK lymphocytes,CD 19+
antigen for B lymphocytes, and CD3+ antigen for T lymphocytes.
The ligase detection reaction (LDR) and ligasechain reaction (LCR)
analyses’ were performed either using a genomic DNA or a cDNA
template prepared by reverse polymerase chain reaction (PCR) from
total cellular RNA.” The LDR assay was performed as described?
The PCR primers for amplification of the genomic DNA were 19mer
corresponding to the nucleotides 1 189-1207 and a 19mer complementing nucleotides 1407-1389. For amplification of the cDNA
nested primers were used. These included a 20mer corresponding to
nucleotides 752-771, and a nested 20mer corresponding to nucleotides
777-796. The reverse outer primer complemented nucleotides 979960, while nested 20mer complemented nucleotides 955-936. The
oligonucleotidesfor LDR were 23mer corresponding to nucleotides
1291-1311 with an additional 5’CT and 3’Cending for the C allele
and a 25mer corresponding to nucleotides 129 1- 13 1 1 with additional
5’ CTTT and 3’ T-ending for the T allele. The ligation partner was
an adjacent common 3’ oligonucleotide,a 2 1mer corresponding to
the nucleotides 1312-1332, labeled with 32Pat 5’ end by T4 kinase.
The LCR analysis used genomic DNAs as templates under conditions
identical to those described previously with additions of the complementary oligonucleotides. The schematicoutline of the experimental
design is outlined in Fig I. To test the usefulness of this assay we
performed analyses of the hematopoietic cells with clonal hemato-
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270
CORRESPONDENCE
genomic DNA
mRNA
rtPCR
+
.1
cDNA
+ PCR +
+ LDR +
I
10% denaturing gel
T
Determination of genotype
Determination of clonality
Fig 1. Schematic outline of the transcriptional analysis of the
assay of the active X-chromosome products using rtPCR,PCR,and
LDR reactions.
poiesis in a patient with myeloproliferative disease and normal hematopoietic cells obtained from random heterozygotes for this polymorphism.
Because this particular polymorphism is not recognizable by any
known restriction enzymes we have used the LDR and the LCR to
detect both alleles of this locus.’ In this assay, the T allele is detected
by a 46-nucleotide fusion product, while the presence of the C allele
results in the synthesis of a 44-nucleotide fusion product. Predictably,
the LCR assay was more sensitive but displayed moderate preference
for the C allele when equimolar amounts of both target DNAs were
present. However, the LDR displayed only minimal preference for
C allele and was therefore used for further studies. In the LDR two
allele-specific oligonucleotides compete for ligation to a common
adjacent oligonucleotide labeled at its 5’ end. Quantitative detection
of both alleles and their easy discrimination was accomplished when
both T and C alleles were present in dilution mixtures ranging in
ratios from 5:95 to 955, respectively. When the level of either allele
in the mixture was I% or less, nonambiguous interpretation of the
data could not be achieved (data not shown). This fact indicates that
a minute contamination of a studied clonal cells by polyclonal inflammatory and other cells would not interfere with the clonality
analyses. The optimal amount of template amplified genomic DNA
and cDNA, using the described experimental conditions, has ranged
from 50 pg to 20 ng (0.3 fmol to 100 fmol).
We tested the validity of the assay by studying the extent of hematopoietic cell involvement in a myeloproliferative disease, polycythemia rubra vera. We studied a 55-year-old white woman whose
polycythemia vera was diagnosed in 1980. Throughout the course of
her disease, the elevated levelsof erythrocytes, granulocyticleukocytes,
and platelets have been present, but no detectable lymphocyte expansion has been noted. As shown in Fig 2, this subject is heterozygous
for the G6PD C T polymorphism, but has only the T-allele transcript
present in circulating erythrocytes, granulocytes and platelets, indicating the clonality of myeloid lineage as previously reported! Circulating B, T lymphocytes, and natural killer (NK) cells have both
transcripts in the ratio of 4 I.
We have screened 19 healthy female volunteers for heterozygosity
for these alleles; seven females were informative for our studies. Their
genomic DNA analysis, and that of the subject with polycythemia
vera (Fig 2). showed an identical ratio of T & C allelic transcripts at
4.65.4, indicating a slight preference of the reaction conditions for
the C allele. The platelet, reticulocyte, granulocyte, and lymphocyte
RNAs from six available heterozygous individuals were isolated using
above described methods. As shown in Fig 3, the ratios ofthese allelic
transcripts in an individual were always remarkably constant in all
hematopoietic terminally differentiated cells and varied widely from
individual to individual, T/C ratio 2:8 to 653.5. These data support
the notion that at the time of X-chromosome inactivation, the only
hematopoietic progenitor is the pluripotent stem cell, which is a precursor of both lymphoid as well as myeloid blood cell lineages. The
results also suggest that more than one pluripotent stem cell is present
at the time of X-chromosome inactivation. The range of C and T
ratios is consistent with more than five cells.
This report describes a highly specific and sensitive assay for the
detection of clonality in cells, cell lineages, and tissues. The assay
based on X-chromosome inactivation is biologically sound because
it discriminatestranscripts of the active X-chromosome. It uses reverse
transcription of mRNA sequences from a polymorphic locus (nucleotide I3 l l, conservative CT mutation in I l exon) of G6PD gene.
The analysis of cDNA by ligase chain reaction makes the detection
of the polymorphism very specific. The high frequency of the nucleotide I3 l l CT polymorphism in all ethnic groups, estimated to
I
I
I
I
I
I
I
I
I
I
I
- 46
- 44
-Fig 2. The LDR analysis’ using a cDNA template prepared by
reverse PCR reaction from total cellular RNA’O of a female with
polycythemiavera. Labeled primer lane contains only the radiolabeled oligonucleotide; neg. control represents sham experiment
without added DNA template; C/Ccontrol representsthe analysis
of DNA from an individual homozygous for the C polymorphism. T/
T control, target DNA from homozygous individual for the T polymorphism and in the C/T control, the target DNA is from a heterozygous individual for the C and T polymorphisms. P, platelets; R,
red blood cells; G, granulocytes; NK, NK lymphocytes; B, B lymphocytes; T, T lymphocytes.
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CORRESPONDENCE
P R G L
27 1
P R G L
P R G L
A
nonhematopoietic tissues that likely represent randomness of the
“Lyonization” process, open a novel approach to the studies of hematopoiesis and embryogenesis in general. The observation of ratios
in hematopoietic tissues suggests that there is only a small number
of undifferentiated hematopoietic cells at the time of X-chromosome
inactivation in the early embryo that further differentiate only after
the X-chromosome inactivation process is completed.
While the preparation of our manuscript was underway, Cumutte
et all2 have independently published in a preliminary form transcrip
tional assay of X-chromosome, using a different detection of C/T
G6PD alleles for the study of a family suffering from chronic granulomatous disease.
ACKNOWLEDGMENT
We are grateful to Dr Ernest Beutler for providing us with homozygous and heterozygous templates for detection of C and T no.
13I I G6PD polymorphisms.
Fig 3. The LDR analysis of purified platelets (P), reticulocytes
(R), granulocytes (G), and lymphocytes (I.)using a cDNA template
prepared by reverse PCR reaction from total cellular RNA from six
normal females heterozygous for GGPD C and T polymorphisms.
(A) Individuals 1 through 3;(6)individuals 4 through 6.
be around 40%: makes the assay imminently suitable for studies of
clonality in neoplastic disorders as well as for embryologic studies.
We have shown the usefulness of the assay by examination of
clonality in seven subjects. The study of polycythemia vera subjects
confirms previously reported clonality of myeloid lineages (red blood
cells, neutrophils, platelets)? The unbalanced T/C ratio ( 4 I ) seen in
lymphoid cells is weighed in favor of the abnormal (T) clone. This
may indicate that lymphoid population is contributed to, in part, by
the PV clone. This explanation would place the original PV mutagenic
event to the pluripotent stem cell common to both myeloid and
lymphoid lineages and it would further suggest that the regulatory
processes governing lymphopoiesis allow normal progenitors to contribute to the generation of circulating progeny, while in myelopoiesis
the proliferation and differentiationderived from the clonal progenitor
is favored. This concept is supported by the fact that in polycythemia
vera both normal and clonal progenitors are present in the bone
marrow, but the clonal progenitors contribute to the bulk of myelopoiesis.” There is an alternate explanation of the 4 1 ratio of T
and C mRNA transcripts observed in all subclasses of circulating
lymphocytes. The possibility cannot be excluded that the mutation
event leading to clonal hematopoiesisoccurred in a more differentiated
hematopoietic progenitor, the myeloid stem cell and then the unequal
T/C ratio in lymphoid cells is caused by the random nature of Xchromosome inactivation. This so-called Lyonization phenomenon
may result in an unequal proportion of alleles forming the mosaic
of female cells, especially in view of the results we obtained in studies
of the normal heterozygotes. This explanation is less likely because
the myeloid stem cell would not be expected to have a self-renewal
potential throughout the 12 years’ duration of the disease in this
patient; however, this may not apply to the myeloid cell modified by
the clonal mutation.
The identical T/C ratios of the lineages of hematopoietic tissue of
each of the six normal heterozygotes studied, taken together with the
wide variationsamong the individualsand with variations seen among
J.T. PRCHAL
Y.L. GUAN
University of Alabama at Birmingham
J.F. PRCHAL
McGill University
Montreal. Quebec, Canada
F. BARANY
Cornell University Medical College
New York. NY
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8. Kay AC, Kuhl W, Prchal JT, Beutler E The origin of glucose6-phosphate-dehydrogenase (G6PD) polymorphisms in AfricanAmericans. Am J Hum Genet 50394, 1992
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1993 81: 269-271
Transcriptional analysis of the active X-chromosome in normal and
clonal hematopoiesis [letter]
JT Prchal, YL Guan, JF Prchal and F Barany
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