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From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1994 84: 2376-2378
Interpretation of X-chromosome inactivation patterns [letter;
comment]
RE Gale and DC Linch
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2376
CORRESPONDENCE
Interpretation of X-Chromosome Inactivation Patterns
To the Editor:
We wish to comment on the recent paper by Fey et al’ using
the M27P probe to detect clonality and X-chromosome inactivation
patterns in hematopoietic cell populations. They p i n t out that the
interpretation of the results in individual cases can be highly problematic, and we are in full agreement with this view. However, we
would take issue with three points raised by these authors. Firstly,
they say that ‘‘M27B discriminates less reliably between active and
inactive alleles than phosphoglycerate kinase (PGK)and hypoxanthine phosphoribsyl transferase (HPRT)” and suggest that “Partial
methylation of inactive X chromosomes . . . probably contributes
to the apparent higher ascertainment of skewing in blood leukocytes
[at the DXS255 locus].” However, the failure to completely digest
either allele with Hpa I1 in hematologically normal or leukemic cell
populations can be iargely overcome by the use of Hhal. and the
results correlate well with those obtained using &aII.* The frequent
skewing of X-chromosome inactivation patterns with M270 in normal blood cells is also seen as frequently with PGK and HPRT.’
Fey et al refer to a major lack of concordance between M27P and
either PGK or HPRT in 5 of 48 samples, but in 3 of 5 samples, the
excess skewing was not observed in the M27P analysis, but was
observed when using PGK. In OUT studies, we observed good concordance between M27P and PGK or HPRT in 46 hematologically
normal leukocyte samples (provided that all digested M270 bands
were included)? Thus,we believe that the skewing of allele digestion
with methylation-sensitive enzymes in normal leukocytes is not a
methodologic artefact with M278, but a true reflection of X-chromosome inactivation.
Secondly, they imply that their data is not compatible with the
Lyon hypothesis and propose an alternative model. We do not be-
lieve this is necessary or helpful. From the Lyon hypothesis, one
would predict that if random X-chromosome inactivation occurred
at a time when the hematopoietic stem cell pool was small, then
skewing would be seen in a significant number of individuals (Fig
1, Table 1). The lesser degree of skewed X-chromosome inactivation
patterns in other tissues found by Fey et al’ and ourselves6may then
reflect a larger stem cell pool size for those tissues at the time of
X-chromosome inactivation. This maymeanthat
commitment to
hematopoiesis has a restricted cell distribution in the embryo at the
time of inactivation’; however, it is worth noting that in the mouse,
X-chromosome inactivation is not complete in all tissues at the same
time: which will similarly influence the patterns obtained.
Thirdly, Fey et a1 refer to skewed patterns as nonrandom, but we
believe that it is precisely because X-chromosome inactivation is
random and occurs in a small stem cell pool that skewing occurs.
If, in an individual with randomly skewed X-chromosome inactivation, a monoclonal tumor arises from a cell with the minor allele
active, and if the tumor population is less than 90% pure, then Xchromosome inactivation ratios well below 10 can be seen. It can
be particularly difficult to ensure such tumor purity in lymphoma
samples where reactive lymphocytes are frequent. Consequently,
suitable controls of normal tissue homologous to the tumor tissue
of interest are not only a way around this problem, as they themselves
point out, but are necessary to interpret X-chromosome inactivation
patterns in individual cases. Fey et al have observed a greater degree
of extreme skewing in leukocytes from normal individuals over the
age of 75 years. In our studies, 4 of 12 (33%) females over the age
of 75 had allelic cleavage ratios (ACR) > 10 in comparison with 4
of 73 (6%) between 20 and 58 years of age! However, it should be
noted that in all four elderly individuals there was extreme skewing
of rhe X-chromosome inactivation patrern in T cells as well as granu-
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2377
CORRESPONDENCE
40
I
I
30 T.
I
25-
0
-
16 cells
8 cells
4 cells
20
3020-
v)
P)
15
(D
a
0
15-
20-
*-
10
0
p
10-
10-
5
5-
0
I
I
0
I
I
I
I
25
I
I
I
I
I
I
I
I
0-
I
75
50
100
0
I
25
0
50
75
100
0
25
50
75
%A
%A
%A
37%' with 25 5 A 2 75%
1 8%' with 25 5 A 2 75%
5%* with 25 5 A 2 75%
1 I
Fig 1. Expected distribution of random X-chromosome inactivation patterns according to stem cell pool size at the time of inactivation.
*This figure representsthe sum total of cases with values <25% or >75% and half of those cases with values equal to 25% or 75%.
locytes, and nonhematopoietic tissues from 2 of these 4, either muscle or skin and muscle, were also found to have imbalanced Xchromosome inactivation patterns. We believe that further studies
in the elderly are warranted. Even if a higher degree of skewing is
confirmed, this might represent selection of only a few clones and
does not mitigate against the Lyon hypothesis.
Rosemary E. Gale
David C. Linch
Department of Haematology
University College London Medical School
tondon, UK
Table 1.
Expected Proportionsof Patients With
Skewed Patterns
~~~
12
No. ofStem Cells at
Time of X-Chromosome
Inactivation
~75%
or 6 2 5 % of
Digested Alleles
(ACR =3l
1
4
8
16
32
100
37
18
5
0.412
~
~91%
or SS% of
Digested Alleles
(ACR 2 1 0 )
100
1
0.044
0.0002
REFERENCES
1. Fey MF, Liechti-Gallati S , von Rohr A, Borisch B, Theilkas
L, Schneider V, Oestreicher M, Nagel S, Ziemiecki A, Tobler A .
Clonality and X-inactivation patterns in hematopoietic cell populations detected by the highly informative M27P DNA probe. Blood
83:931, 1994
2. Gale RE, Linch DC: Investigation of methylation at Hha I sites
using the hypervariable probe M27P allows improved clonal analysis
in myeloid leukaemia and demonstrates differences in methylation
between leukaemic and remission samples. Leukemia 8:190,1994
3. Gale RE, Wheadon H, Linch DC: X-chromosome inactivation
patterns using HPRT and PGK polymorphisms in haematologically
normal andpost-chemotherapy females. Br J Haematol79:193,1991
4. Gale RE, Wheadon H, Linch DC: Assessment of X-chromosome inactivation patterns using the hypervariable probe M27P in
normal hemopoietic cells and acute myeloid leukemic blasts. Leukemia 6549, 1992
5. Fey MF, Peter HJ, Hinds HL, Zimmermann A, Liechti-Galati
S , Gerber H, Studer H. Tobler A: Clonal analysis of human tumors
with M27P, a highly informative polymorphic X-chromosomal
probe. J Clin Invest 89:1438, 1992
6. Gale RE, Wheadon H, Boulos P, Linch DC: Tissue specificity
of X-chromosome inactivation patterns. Blood 83:2899, 1994
7. McLaren: Numerology of development. Nature 239:274, 1972
8. Tan S S , Williams EA, Tam PPL: X-chromosome inactivation
occurs at different times in different tissues of the post-implantation
mouse embryo. Nat Genet 3:170, 1993
Response
We would like to thank Dr Belmont as well as D r S Gale and
Linch for their interesting comments on our work on clonality and
M27P X-inactivation patterns in hematology.' Dr Belmont suggests
that our way of expressing the results of M27P X-inactivation analysis by calculating allelic cleavage ratios would lead to a distorsion
of allelic cleavage ratio (ACR) values towards very large numbers
as allelic contribution gets increasingly skewed.'.2 For practical reasons we have set the limits of the ACRs at l (lower end representing
random X inactivation) and at 100 (upper end representing 100%
unilateral use of one X chromosome) as outlined in Fig 2 of our
paper.' As a corollary, an extreme distorsion of the results would
be avoided. However, data compiled in this way were not normally
distributed, and hence, nonparametric tests were chosen for statistical
analysis. We share Dr Belmont's concern that our observations seem
to undermine the correlation between X-chromosome activation status and particular methylation patterns at the M27P locus. His find-
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
2378
ings of mismatches between results of M278 analysis and clonal
analysis using the highly informative androgen receptor locus (HAR)
are of particular interest in this r e ~ p e c tWe
. ~ do not as yet have full
results of HAR-X-inactivation analysis in our cases. However, our
own preliminary data suggest that indeed correlation between clonal
HAR and M278 analyses might not beconsistent in all cases (unpublished results). When comparing various approaches to study clonal
X inactivation, we find it difficult to judge at this stage whether data
obtained with M278 X-inactivation analysis are more likely to be
inaccurate than results obtained with the phosphoglycerate kinase
andthe hypoxanthine phosphoribosyl transferase gene polymorphisms, the HAR marker, or the glucose-6-phosphate-dehydrogenase
protein variants. Unfortunately, the gold standard reflecting the true
X-inactivation pattern of a particular case has notbeen defined.
Therefore, we would certainly agree with Dr Belmont’s warning,
which is also expressed in our paper, that results of M278 clonality
assessment should be interpreted with great caution in hematologic
or any other samples ifno homologous normal tissue control is
available, because extreme skewing might be constitutional rather
than reflecting the clonal neoplastic nature ofa cell population.
Unfortunately, such control material is rarely if ever athand in
hematologic cases. We have offered a few speculative explanations
in our paper as to why skewedX inactivation might be more frequent
at the M278 locus than at others, and Dr Belmont has thoughtfully
extended our discussion on this interesting point. Although we cannot offer any formal proof, we still favor the hypothesis of a clonal
succession model in hematopoiesis to explain age-related skewing
in our patient population and we believe that Dr Belmont’s discussion of our findings may be taken as favoring the same basic idea.
Drs Gale and Linch suggest that the problem of discriminating
between active and inactive alleles at M27P maybe overcome by
using Hha I instead of Hpa 11. We are grateful for this helpful
comment and we are indeed currently testing our series of samples
with Hha I. In a second comment, they p i n t out that the smaller
the number of hematopoietic stem cells at the time of X inactivation,
the higher the chances are for observing constitutionally skewed X
inactivation inblood cells. If, for example, hematopoiesis would
depend on a single stem cell, severe skewing would be seennot
only in a clonal leukaemia from such an individual but equally in
her normal leukocyte DNA. Assessment of clonality in such patients
would obviously be problematic. Similarly, the degree of constitutive
skewing in hematologic cell populations would increase over time
if, with advancing patient age, hematopoiesis would depend on fewer
and fewer actively dividing stem cells producing mature blood cells.
Our data would support this notion. However, there isnoway to
CORRESPONDENCE
determine in a given normalindividual whether skewing at theM270
locus is caused by small size of the original hematopoietic stem cell
pool present at thetime of lyonization or whether a clonal succession
model might be responsible. Based on our observation of an increasingrate of skewing with advancing ageofthepopulationunder
investigation, we believe that both ideas must be considered. However, it was never our intention, nor is it our current view that our
data should be read as overthrowing Dr Mary Lyon’s X-inactivation
hypothesis, andwe feel that this should appear clearly from our
model shown in Fig 4.’ We were very interested to note that in their
small series of elderly women, Gale et a14 were able to confirm our
finding of an increased rate of severe skewing in blood leukocytes.
We absolutely agree with their point that this phenomenon would
warrant further investigation and we have never pretended that this
finding, if confirmed, would mitigate in any way against the Lyon
hypothesis.
In summary, webelievethat although the M278 assay i s very
usefulto assess clonality in solid tumors, its limitations for this
purpose in hematology are such that its use should be discouraged.
Martin F. Fey
Albert von Rohr
Institute of Medical Oncology
Andreas Tobler
Central Haematology Laboratory
University of Berne
Berne, Switzerland
REFERENCES
1. Fey MF, Liechti-Gallati S, von Rohr A, Borisch B, Theilkas
I, Schneider V, Oestreicher M, Nagel S, Ziemiecki A, Tobler A:
Clonality and X-inactivation patterns in hematopoietic cell populations detected by the highly informative M278 DNA probe. Blood
83:931, 1994
2. Fey MF, Peter HJ, Hinds HL, Zimmermann A, Liechti-Gallati
S, Gerber H, Studer H, Tobler A: Clonal analysis of human tumours
with M278,a highly informative polymorphic X-chromosomal
probe. J Clin Invest 89:1438, 1992
3. Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont
JW: Methylation of Hpa I1 and Hha I sites near the polymorphic
CAG repeat in the human androgen receptor gene correlates with X
chromosome inactivation. Am J Hum Genet 51:1229, I992
4. Gale RE, Wheadon H, Boulos P, Linch DC: Tissue specificity
of X-chromosome inactivation patterns. Blood 83:2899, 1994