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Skewed X-Inactivation in Carriers of X-Linked Dyskeratosis Congenita
By T.J. Vulliamy, S.W. Knight, I. Dokal, and P.J. Mason
A gene causing Dyskeratosis Congenita (DC), a rare genetic
disorder associated with bone marrow failure, has been
mapped to chromosome Xq28, but autosomal inheritance
of the disease has also been reported. We have investigated
the pattern of X-inactivation in the peripheral blood of carriers of DC using the methylation-sensitive Hpa II site in the
androgen receptor gene (HUMARA). In 5 different families
in which the inheritance of DC appears to be X-linked, all 16
carriers showed skewed X-inactivation patterns. These
cases indicate that, in the hematopoiesis of heterozygous
females, cells expressing the normal DC allele have a growth
advantage over cells that express the mutant allele. In 7
other families with sporadic cases of DC or with an uncertain
pattern of inheritance, both skewed and normal patterns
of X-inactivation were observed. In these families or where
crucial family members are unavailable, the study of X-inactivation patterns will add to linkage analysis in providing
information about carrier status.
q 1997 by The American Society of Hematology.
002, 005, 006, and 007). In 4 of the remaining 7 families, there is
only a single affected male known to us (families 004, 008, 015,
and 020). In 1 family (019), the father is affected and one of his
daughters has aplastic anemia, but no other features of DC. In another
family (024), there is an affected male and an affected female in
one generation. In the remaining family, which has been described11
(family 009), there is one boy who is clearly affected with DC and
he has two male first cousins who died very young from bone marrow
failure. For the 25 relevant females, DNA was obtained from a whole
blood sample by conventional phenol-chloroform extraction.12
Analysis of X-inactivation. X-inactivation was analyzed using
the methylation-sensitive Hpa II site located near the highly polymorphic trinucleotide repeat in the human androgen receptor gene
(HUMARA)13 in Xq11.2-Xq12. Amplification was performed after
or without Hpa II digestion with primers flanking the CAG repeat
and the Hpa II site. After Hpa II digestion, no amplification product
is seen from the active X-chromosome, because the site is unmethylated and cleaved. Amplification products were analyzed on standard
6% sequencing polyacrylamide gels containing 8 mol/L urea. The
only modification we have made to the previously described
method13 was a reduction of the number of polymerase chain reaction
cycles from 28 to 25. The dilution of amplified DNA in formamide
loading dye was sometimes adjusted to even out the DNA load.
Quantification of autradiographs was performed at short exposure
times (õ 16 hours) using a UVP camera system (GDS7600; Ultra
Violet Products Ltd, Cambridge, UK) and the NIH Image/68k 1.57
program (Wayne Rasband [[email protected]], National Institutes of Health, Bethesda, MD).
YSKERATOSIS CONGENITA (DC) is a rare inherited
disorder associated with bone marrow failure and a
predisposition to malignancy. Although it is not always easy
to make the diagnosis of DC, the disease is characterized
by nail dystrophy, mucosal leukoplakia, and reticulate skin
pigmentation.1-3 DC is often inherited as an X-linked recessive disorder (DC, MIM 305000), and in one large pedigree,
Connor et al4 mapped the DC gene to Xq28. This linkage
has been confirmed in three additional families,5 and we
have recently mapped the DC locus to 3.8 Mb of DNA
within this region,6 although the gene itself has not yet been
There is also evidence that the disease is heterogeneous,
and autosomal dominant and recessive modes of inheritance
have been recognized (MIM 127550 and MIM 22430). Although we have studied a number of multiplex families in
which the disease is present, 16 of the 29 families that have
been referred to us are sporadic, with only 1 family member
affected. Although most of these cases are male and therefore
consistent with an X-linked mode of inheritance, genetic
counselling of family members is difficult in view of the
heterogeneity of this disorder.
Skewed X-inactivation has been observed in X-linked disorders such as X-linked agammaglobulinemia,7 incontinentia
pigmenti type 2,8 and Wiscott-Aldrich syndrome, in which
skewing has been used as a method for carrier detection.9
In this study, we show that skewed X-inactivation is also
seen in female carriers of DC in 5 different families in which
the disease is clearly inherited as an X-linked disorder, consistent with a previous report on 1 X-linked family and the
mother of a single sporadic case.10 We have also extended
the analysis to our unique collection of sporadic families
with DC to see if this is a consistent finding and to see if
X-inactivation analysis can help us to recognize the X-linked
form of the disease and to determine the carrier status of
female family members.
Families and sample preparation. Families with DC were obtained through the DC registry established at the Hammersmith Hospital. In each family, the diagnosis of DC was made when the index
case had the three following muco-cutaneous features by the age of
15 years: (1) skin pigmentation, (2) nail dystrophy, and (3) mucosal
leukoplakia. Twelve families could be included in this study because
fresh blood samples had been referred from female relatives of the
index case, and these females were informative for the HUMARA
polymorphism (see below). They are shown in Fig 1. Five of these
families have been previously described,3-5 and in these, linkage of
the disease to markers in Xq28 has been established (families 001,
X-linked families. In the 5 families in which we have
shown linkage of the DC gene to markers in Xq28, all of
the 16 female carriers analyzed show skewed X-inactivation
patterns (Table 1 and Fig 2). This is true for both the obligate
carriers as well as for those that we predict to be carriers
based on linkage analysis. In 2 of these females (family 005,
From the Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Submitted February 7, 1997; accepted May 8, 1997.
Supported by the MRC (UK) and through funds from the Department of Haematology, RPMS, London, UK.
Address reprint requests to T.J. Vulliamy, PhD, Department of
Haematology, Royal Postgraduate Medical School, Hammersmith
Hospital, Ducane Road, London W12 ONN, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
Blood, Vol 90, No 6 (September 15), 1997: pp 2213-2216
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Fig 1. DC families. The carrier status of women marked with an asterisk has been inferred from their X-inactivation status. The shaded
individuals have aplasia but do not show the dermatologic features of DC.
III:3 and family 006, II:4), the skewing is not complete, and
a faint residual band can be seen for one allele (eg, Fig 2,
lane 12). From the densitometry plots (Fig 3A and B), we
estimate that the minor band in these cases represents 5.5%
of the total signal in the obligate carrier (006, II:4) and 3%
in the predicted carrier (005, III:3).
In 3 of these families in which we have analyzed the
appropriate fathers, we can show that it is the paternal allele
Table 1. X-Inactivation Patterns in Carriers of DC
Pattern of X-Inactivation*
X-linked, obligate carrier
X-linked, predicted carrier
Sporadic, mother of index case
Sporadic, daughter of index case
Sporadic, predicted carrier
Familial, mother of index case
Uncertain pattern of inheritance
Incompletely Skewed
II:5 and III:2
I:2 and II:3
II:6 and II:11
II:7, III:4, and III:7
015, II:1
006, II:4
005, III:3
015, I:1
020, I:1
019, II:1
024, I:1
009, II:1 and II:12
* X-inactivation patterns were scored as skewed when, after Hpa II digestion, only one allele of the HUMARA gene from an informative female
was visible or when one allele represented less than 2% of the total signal. The pattern was scored as incompletely skewed when the faint
allele represented 2% to 10% of the total signal.
† The identity of each female is given by family number and position as shown in Fig 1.
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Fig 2. Examples of X-inactivation analysis. Amplification by polymerase chain reaction across the HUMARA polymorphism was performed as described
in the Materials and Methods. Lanes marked f show
the allele amplified from the father of the female
studied in the next two lanes; Ï is without Hpa II
digestion and " is with Hpa II digestion before amplification. Lanes 2, 3, and 5 through 12 show females
from X-linked families. In these samples note the
skewed inactivation patterns, although lane 12 is incompletely skewed. Lanes 13 and 14 show a normal
pattern in the mother of family 024.
that fails to amplify after Hpa II digestion (eg, Fig 2, lanes
1 through 6). Therefore, it is the maternal chromosome, on
which the mutated DC gene is carried, that is always methylated and inactive.
Relatives of sporadic cases. In 5 of the families investigated, there is only a single affected male. Three of the
mothers of these patients (in families 004, 008, and 015)
show skewed X-inactivation patterns, although in one of
them (family 015) this skewing is incomplete (Table 1). A
sister of a sporadic case (family 015, II:1), who is predicted
to be a carrier by linkage analysis, also shows a skewed
pattern. However, 1 mother (in family 020) shows a random
pattern of X-inactivation. In the fifth sporadic family (019),
the affected male has two daughters who would be obligate
carriers if the DC was X-linked in this family: one daughter
has aplastic anemia but is uninformative for the HUMARA
polymorphism, but her sister (019, II:1) shows a random
pattern of X-inactivation.
Familial DC, probably unlinked to Xq28. In family 024,
there is an affected female and an affected male in one
generation. Their mother shows a random pattern of X-inactivation (Fig 2, lanes 13 and 14). In 1 previously published
family11 (009), a single boy is clearly affected with DC. He
has two male cousins that died very young with bone marrow
Fig 3. Densitometry plots of X-inactivation analysis. Individual
lanes showing amplification of the HUMARA gene after Hpa II digestion are plotted using the NIH image program. Autoradiographs were
exposed for less than 16 hours. (A) The incompletely skewed female
shown in lane 12 of Fig 2 (family 6, II:4). (B) The other incompletely
skewed female from an X-linked family (family 005, III:3). (C) The
mother of an affected boy from the family with an uncertain pattern
of inheritance (family 009, II:12). (D) Her sister (family 009, II:1).
Blood 0023
failure, and so it is proposed that these boys also had DC;
the inheritance therefore appears to fit an X-linked pattern.
However, in this family, the disease gene does not appear
to be linked to markers in Xq28 (data not shown). Although
slightly skewed, both of the relevant mothers in this family
(009, II:1 and II:12) have X-inactivation patterns within the
normal range:7,14 the relative intensities of the minor bands
are measured at 33% (Fig 3C) and 18% (Fig 3D) in densitometry plots.
In this study we have shown that of 16 females that were
obligate or predicted carriers of X-linked DC all showed
skewed X-inactivation patterns. Although skewing may be
observed in approximately 9% of normal subjects15 and older
women in general are more likely to show a skewed pattern,16
the consistency and completeness with which it is observed
in these carriers indicates that it could not have occurred by
chance alone. At an early stage in development in female
mammals, either the paternal or maternal X chromosome is
inactivated at random in each cell. Therefore, the finding of
preferential inactivation of the DC X chromosome in blood
cells of female DC carriers indicates that cells expressing
the normal allele at this locus have a growth or survival
advantage over cells that express the mutated allele. This
finding supports the idea that the product of the DC gene
plays an important role in hematopoiesis. It is also consistent
with our previous observation that the growth and proliferation of DC fibroblasts is abnormal.17
Two of the carriers, one of whom is a 15-year-old girl,
did not show complete skewing. In affected males, the age
at which symptoms of DC develop can vary considerably,
presumably due to different mutations, and it is possible that
this is reflected in the age and the degree to which skewed
X-inactivation is manifested in female carriers. It may be
appropriate to test other cells of young female carriers such
as fibroblasts, because if a certain number of cell divisions
are required before the putative growth advantage is manifested, fibroblasts may reach this state before lymphocytes.
A previous report has also shown highly skewed X-inactivation patterns in a single X-linked family with DC and in
the mother of one sporadic case.10 The consistency of this
finding that we have seen here in 5 other families with the
X-linked form of DC allows us to extend the analysis to
study the relatives of sporadic cases of the disease. Although
the likely form of inheritance of DC in most of these families
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is as an X-linked recessive trait, it is possible that the inheritance is autosomal or that the index case is the product of a
de novo mutation. We have found that, of 4 tested, 3 mothers
of different sporadic cases of DC (families 004, 008, and
015) showed a skewed pattern of X-inactivation. We believe
that this information helps us in counselling the families,
allowing us to be more confident of the predictions made
through linkage analysis using Xq28 markers, and even enables us to search for recombination events if there are informative meioses.
We have also seen random X-inactivation patterns in 2
mothers (families 020 and 024) and 1 daughter (family 019)
of unrelated patients with DC. In these families, we can
suggest that the disease is either not X-linked or that there
is a de novo mutation. In 1 family (009) in which we are
uncertain as to the pattern of inheritance of the disease, we
find only slightly skewed patterns of X-inactivation that
could be considered to fit within the normal range. If the
disease is indeed X-linked in this family, then this would
confound our hypothesis that the carriers of the X-linked
form of DC consistently show highly skewed patterns, although it is also possible that the disease in this family is
caused by another X-linked gene that is not the DC gene in
Xq28, and this does not produce skewed inactivation in female carriers. It is clear that some of the suggestions made
here will only be confirmed or refuted when the DC gene
in Xq28 has been cloned, enabling direct mutation analysis.
On the basis of X-inactivation studies it has been suggested that a large proportion of patients with acquired aplastic anemia exhibit clonal hematopoiesis,18,19 although other
studies find clonal hematopoiesis in a smaller number of
such patients.20 Our results suggest that the relationship between X-inactivation and clonal hematopoiesis may not be
so straightforward. Specifically, if the expression of any Xlinked gene is defective in aplasia, polyclonal hematopoiesis
may appear to be monoclonal, owing to a skewed X-inactivation pattern resulting from the growth or survival advantage
of all cells expressing the nondefective X chromosome.
In conclusion, the studies presented here, along with other
recent findings,10 now provide sufficient evidence to suggest
that the X-inactivation assay can be used with some confidence to determine carrier status in potential female carriers
of X-linked DC. In addition, we have shown that the assay
can be useful in making a diagnosis of X-linked DC in
sporadic cases by testing the mothers and/or sisters of affected individuals, enabling us to counsel these families appropriately. However, it should be borne in mind that some
skewing can occur in normal females and that in young
female carriers skewing may be incomplete.
The authors thank the members of participating families and the
doctors who have referred samples to us: Prof B. Alter, Dr A. Copplestone, Dr G.L. Forni, Dr A. Fryer, Prof E. Gluckman, Prof R.E.
Hall, Dr G. Lowe, Dr D. Oscier, Prof J.A. Raeburn, Dr I. Roberts,
Dr J. Ross, and Dr O. Smith. The authors are also grateful for the
support of Prof J.M. Goldman.
Blood 0023
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1997 90: 2213-2216
Skewed X-Inactivation in Carriers of X-Linked Dyskeratosis Congenita
T.J. Vulliamy, S.W. Knight, I. Dokal and P.J. Mason
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