Epigenetics in Turner syndrome
Nava and Lanes Clinical Epigenetics
Epigenetics in Turner syndrome
Francisco Álvarez-Nava 0
Roberto Lanes 1
0 Biological Sciences School, Faculty of Biological Sciences, Central University of Ecuador , Quito , Ecuador
1 Pediatric Endocrine Unit, Hospital de Clínicas Caracas , Caracas , Venezuela
Background: Monosomy of the X chromosome is the most frequent genetic abnormality in human as it is present in approximately 2% of all conceptions, although 99% of these embryos are spontaneously miscarried. In postnatal life, clinical features of Turner syndrome may include typical dysmorphic stigmata, short stature, sexual infantilism, and renal, cardiac, skeletal, endocrine and metabolic abnormalities. Main text: Turner syndrome is due to a partial or total loss of the second sexual chromosome, resulting in the development of highly variable clinical features. This phenotype may not merely be due to genomic imbalance from deleted genes but may also result from additive influences on associated genes within a given gene network, with an altered regulation of gene expression triggered by the absence of the second sex chromosome. Current studies in human and mouse models have demonstrated that this chromosomal abnormality leads to epigenetic changes, including differential DNA methylation in specific groups of downstream target genes in pathways associated with several clinical and metabolic features, mostly on autosomal chromosomes. In this article, we begin exploring the potential involvement of both genetic and epigenetic factors in the origin of X chromosome monosomy. We review the dispute between the meiotic and post-zygotic origins of 45,X monosomy, by mainly analyzing the findings from several studies that compare gene expression of the 45,X monosomy to their euploid and/or 47,XXX trisomic cell counterparts on peripheral blood mononuclear cells, amniotic fluid, human fibroblast cells, and induced pluripotent human cell lines. From these studies, a profile of epigenetic changes seems to emerge in response to chromosomal imbalance. An interesting finding of all these studies is that methylation-based and expression-based pathway analyses are complementary, rather than overlapping, and are correlated with the clinical picture displayed by TS subjects. Conclusions: The clarification of these possible causal pathways may have future implications in increasing the life expectancy of these patients and may provide informative targets for early pharmaceutical intervention.
Aneuploidy; Chromatin; DNA methylation; Embryonic stem cells; Epigenetics; Gene expression; Mouse models; Turner syndrome
Over 80 years ago, Turner [
] first described a clinical
picture that would subsequently carry his name. Since
then, a great wealth of knowledge has been accumulated
regarding the monosomy of sex chromosomes. It was
] who settled the cytogenetic basis and
FergusonSmith who proposed the existence of genes in the short
arms of both sex chromosomes that would determine
the Turner syndrome (TS) phenotype [
]. From there
on, the cytogenetic findings in patients with this disorder
have been linked to molecular sources allowing us to
better understand its pathogenesis. The girls with TS
have meanwhile grown up, and in recent years, a new
phenotype for this old condition, related to its morbidity
and mortality, has been outlined. Although researchers
have tried to explain the clinical findings in TS based on
cytogenetic and gene changes, everything seems to
indicate that other causes for this disorder, beyond the
nucleotide sequence of a gene or the global impact of
the chromosomal imbalance, exist. These modifications
need to be explored, beginning with the primary etiology
that causes the inadequate segregation of the sex
chromosomes, up to the metabolic disorders that appear
in adult life and which tend to diminish the life
expectancy of women with TS. Similarly, from this
perspective, short stature, gonadal dysgenesis, and
congenital heart disease, as well as intrauterine growth
retardation (IUGR), epidemiologically related to
metabolic problems in other disorders, must be examined.
Evidence collected in recent years does not seem to
indicate only one proposed mechanism to explain the
clinical findings and the associated complications in TS,
but instead the concurrence of several of them. In
addition, there does not appear to be an association
between the primary cause that determines the aneuploidy
of the sex chromosomes and the clinical findings. A
unifying hypothesis to help explain the findings in the
embryonic, fetal, neonatal, and adolescent periods, as well
as in adulthood, and the pathogenesis of this disorder,
relates to changes in DNA conformation and its assembly
without modifying its nucleotide sequence. This review
describes the latest known facts on the role of epigenetics
in the etiopathogenesis of TS. We are now in a stage in
which TS transcends its clinical, cytogenetic, and gene
aspects into the epigenetic landscape.
Although it has been known since 1959 [
] that TS is
caused by a complete or partial deletion of the second
sex chromosome, the mechanisms by which monosomy
of the X chromosome disrupts development is still not
well understood. Several deleted genes from the X
chromosome are expected to largely affect various tissues,
organs, and systems during embryonic development,
growth, and adult life. This hypothesis is known as the
“Gene Dosage Effect,” which explains the cause of TS
based on the absence of a limited number of dosage
sensitive genes localized on the Xp chromosome [
other words, this hypothesis states that developmental,
clinical, or metabolic features of TS can be mapped to
specific regions of sex chromosomes. Thus, based on this
hypothesis, the TS phenotype is a direct consequence of
the cumulative effect of the absence of individual loci
(haploinsufficiency) located on the Xp chromosome [
The Gene Dosage Effect hypothesis is mainly supported
by rare cases of partial Xp deletion, linking TS phenotypes
to a small number of loci in specific segments of the Xp
chromosome. However, genes do not operate as
independent units in the genome but are rather embedded in
temporally and spatially highly coordinated regulatory
networks. Thus, the “Reductionist” view contrasts with
the “Organicistic” view or “Amplified
DevelopmentalInstability” hypothesis which is basically based on
Waddington’s view. It states that the pathogenesis of TS is
strictly connected to the presence of a monosomy of the
second sex chromosome that profoundly disturbs
genomic homeostasis, i.e., the TS phenotype is the result
of global genomic imbalance, rather than the addition of
the effects of individual loci [
]. Nevertheless, recent
research inclines us to support a synthesis of these two
views, which indicates that with a deleted gene, the
individual effect is modest and one could therefore only
explain the TS phenotype by combination with other loci,
based on the specificity of effects and interactions of these
genes . Thus, the TS phenotype may not merely be due
to genomic imbalance from deleted genes linked to the
second sex chromosome but may also be due to additive
influences on associated genes within a given gene
network with an altered regulation of gene expression
triggered by epigenetic factors.
Understanding the role of genetics in establishing
epigenetic patterns has become a high priority in the
postgenomic era. Epigenetic mechanisms include all the
processes involved in creating instructions for a cell to
function efficiently. To carry out this action, epigenetic
machineries perform an important role in controlling
gene expression and, thus, regulating normal and
abnormal cellular processes associated with diseases.
Pathological conditions occur when epigenetics become
decontrolled. Most evidence suggests that epigenetic
modifications are carried out by four mechanisms: (1)
DNA methylation of CpG sites in the promoter region
of the gene, (2) posttranslational covalent modifications
of histones (histones code) and the use of variant
histone proteins, (3) remodeling of nucleosomes and/or
reorganization of chromatin on a larger scale, and (4)
regulation of gene expression by small, noncoding RNA
molecules (microRNA, miRNA). These modifications
are reversible, changing the chromatin configuration
(open or closed), activating or silencing genes, and do
not occur in isolation, i.e., the overall effect is obtained
by the combination, type, site, and extent of modifications.
Increasing evidence demonstrates that epigenetics has a
powerful impact on normal cognition, cardiovascular
development, growth, and lipid and glucose metabolism
]. However, epigenetic effects have been poorly
investigated in TS as, up to now, most of the TS research
has focused on clinical, chromosomal, and genetic
abnormalities (Reductionist view).
Hypothetically, the haploinsufficiency of loci on the
Xp chromosome results in a 0.5-fold diminished gene
expression. However, transcriptome analyses in different
human tissues and in XO mouse models did not reveal a
direct correlation between genomic imbalance and gene
expression levels [
]. Significantly, changes in gene
expression have been also detected in loci that are not
on the X chromosome and diverge between cell and
tissue types [
]. It is important to emphasize
that many genes display broad expression variation and,
even for genes that are deregulated in TS, there is an
extensive overlap of expression levels between 46,XX
and 45,X cells [
]. Thus, it is reasonable to assume
that this variation in gene expression significantly
contributes to the phenotypic variation seen in TS
individuals. In addition, copy number variations might also
explain such variable expressivity in TS . Specific
clinical features exhibit a broad spectrum in TS subjects,
even in subjects with 45,X karyotype. Thus, copy
number variations might help explain such variable
expressivity and be implicated in neurodevelopmental
disorders, thoracic aortic aneurysms, and dissections in
]. However, it seems premature to conclude
whether copy number variations are an influencing
factor in the TS phenotype. On the other hand, the
functional consequences of epigenetic changes, which
occur at a higher rate than DNA sequence changes, are
likely underestimated in this chromosomal disorder.
While haploinsufficiency of genes on the X chromosome
has been the focus of recent research, underlying epigenetic
mechanisms have been poorly studied in TS. Nevertheless,
it is has become clear that epigenetic processes are altered
in TS [
], so that by modulating gene expression,
epigenetics could play a crucial role in altered growth and
in the development of abnormalities of lipid and glucose
metabolism associated with TS. While this has not been
clearly demonstrated so far, it could assist in deciphering
the epigenetic regulation of human IGF1 gene expression,
which would be useful in the diagnosis of growth and bone
metabolism alterations and for the management and
rational use of rhGH therapy. Analyzing the status of DNA
methylation in the PPARGC1A promoter and the biogenesis
of mitochondria and their relevance on the development of
metabolic abnormalities may provide new biomarkers that
could predict cardiometabolic risk in women with TS (see
the “Summary and future perspectives” section).
Due to recent advances in high throughput screening
of both mRNA expression and DNA methylation as a
resource to study epigenetic mechanisms, an increasing
wealth of knowledge regarding gene expression has been
emerging. These recent advances have allowed us to
understand the essential connection between gene
clusters in effecting specific pathways. This review
begins by exploring the potential involvement of both
genetic and epigenetic factors in the origin of X
chromosome monosomy. Several in vivo and in vitro studies
have examined the effects of epigenetic alterations on
the remodeling of chromatin, which disturb the ability
of the chromosomes to align, attach to mitotic spindle
fibers, and/or separate. These studies have led to the
identification of pathways affected in non-disjunction
chromosomal conditions, such as Immunodeficiency,
Centromeric instability and Facial anomalies (ICF) and
Mosaic Variegated Aneuploidy syndromes. We also
review the dispute between the meiotic and post-zygotic
origins of the 45,X monosomy. The meiotic epigenetic
landscape of sex chromosomes is faced with the
chromosomal instability of blastomeres in early
embryonic development, mediated by post-zygotic epigenetic
reprogramming in order to explain the high frequency
of X-monosomy in human conception. Subsequently, we
focus on the phenotypical consequences of epigenetic
modifications in 45,X monosomy, as a profile of epigenetic
changes that seem to emerge in response to chromosomal
imbalance caused by the absence of the second sex
chromosome. This epigenetic pattern may contribute to
both the clinical picture and the phenotypic variation
present in TS patients. We will also discuss the debate
that exists between Reductionists and Organicistic,
through which gene expression regulated by epigenetic
mechanisms allows us to establish a synthetic view of the
clinical picture and the phenotypic variability of TS. Thus,
we analyze the findings from several studies that compare
gene expression of 45,X monosomy to their euploid and/
or 47,XXX trisomic cell counterparts on peripheral blood
mononuclear cells, amniotic fluid, human fibroblast cells,
and induced pluripotent human cell lines. We also review
studies that have provided evidence of an altered
methylome in 45,X fibroblast cell lines, as well as in peripheral
blood mononuclear cells. These in vitro results have been
corroborated in vivo by analyzing the methylation profile
in a well-characterized cohort of women with 45,X
]. An interesting finding of all these studies is
that methylation-based and expression-based pathway
analyses are complementary, rather than overlapping, and
are correlated with the clinical picture displayed by TS
subjects. The clarification of these possible causal
pathways may have future implications in increasing the life
expectancy of these patients, as epigenetics could
potentially pinpoint central avenues of research for the
management and treatment of short stature, bone disorders,
metabolism, and cardiovascular diseases in this condition.
Epigenetic signatures such as histone modifications and
DNA methylation are reversible, unlike the monosomy of
the second sex chromosome, and, thus, offer an enormous
potential in reducing the effects of the chromosomal
imbalances seen in TS. Studies currently being carried out
suggest that changes in DNA methylation in TS patients
may influence lipid metabolism and mitochondrial
biogenesis (unpublished data). These ongoing studies may
provide informative targets for early pharmaceutical
intervention, so as to ameliorate alterations that confer an
increased risk of cardiovascular and cerebrovascular
diseases to patients with TS. Lastly, we discuss how the
haploinsufficiency of Xp chromosome genes may cause a
number of epigenetic consequences that may affect the
appearance of clinical features, associated complications,
and phenotypic variability in TS.
Epigenetic origin of the 45,X monosomy
It has been speculated that several chromosomal
heteromorphisms in certain heterochromatic regions can
directly affect chromosomal malsegregation, possibly by
compromising the fidelity of the spindle attachments
and replication/pairing processes [
]. Large blocks of
heterochromatin between homologous chromosomes
could lead to asynchronous replication, which, in turn,
may contribute to misalignment and nondisjunction [
]. Additionally, the role of heteromorphisms in
chromosomal nondisjunction could be due to disturbances in
recombination, which, in turn, could lead to aneuploidy
]. However, the influence of chromosomal
heteromorphisms appears to be minimal or to act through
complex mechanisms (see below), and its association with
aneuploidy has not been consistently observed.
More than 100 genes could provoke defects in
chromosomal segregation when they are mutated, which
may lead to mitotic spindle alterations, centromere
amplification, cell cycle checkpoint defects,
nonseparation of sister chromatids, and telomere [
However, current evidence points out that such
mutations are uncommon in aneuploidy cells [
Thus, chromosomal behavior may be affected by other
mechanisms that do not include chromosomal
heteromorphisms or gene mutations.
Epigenetic mechanisms may serve as a potential link
for explaining the two hypotheses mentioned above
regarding chromosomal malsegregation [
]. For example,
epigenetic alterations in pericentromeric heterochromatin
may lead to remodeling in chromatin conformation which
may compromise the ability of the chromosomes to align,
attach to mitotic spindle fibers, and/or separate [
This latter hypothesis is based on analyses of
hypomethylated cells obtained either from cells treated with
DNAmethyltransferase inhibitors (5-azacytidine) or from cells
from patients with ICF syndrome who have mutations in
the DNA methyltransferase 3b gene (DNMT3B). This gene
encodes a DNA methyltransferase which is thought to
function in de novo methylation, rather than in
maintenance methylation which is an epigenetic mark.
These analyses showed delays in centromere separation
that led to aneuploidies . Thus, hypomethylation in
whole chromosome (for example in an X chromosome)
might cause chromosomal instability and large-scale
chromosomal changes. Consequently, DNA methylation
and histone modification have an impact on the correct
chromosomal segregation. On the other hand, genes
involved in spindle checkpoint control could not be
mutated but are instead aberrantly silenced in aneuploidy
cells by epigenetic signature [
]. Thus, epigenetic
mechanisms can replace mutations as a way of silencing the
spindle checkpoint gene. Therefore, abnormal
chromosomal segregation can be originated by alterations in DNA
methylation on spindle checkpoint genes.
Although the role of epigenetic changes on the initiation
of malsegregation of sex chromosomes is still largely
speculative, a profile of epigenetic changes seems to
emerge in response to chromosomal imbalance caused by
45,X monosomy, which may not only contribute to the
clinical picture present in TS patients but can also be
associated to malsegregation of sex chromosomes. Thus, a
genome-wide hypomethylation has been described in
leukocytes from 45,X patients [
], which might reactivate
cryptic site transcription start and cause changes in
expression of isoform transcripts. Applying analysis of
differential exon usage to the autosomes in 45,X cell lines,
eight protein coding and two non-coding RNAs genes
differentially spliced have been described [
]. Of these
genes, the BB1 gene is striking since it encodes a kinase
involved in spindle checkpoint function and chromosome
]. The BB1 gene has been localized to the
kinetochore and plays a role in the inhibition of the
anaphase-promoting complex/cyclosome (APC/C), delaying
the onset of anaphase and ensuring proper chromosome
segregation. Impaired spindle checkpoint function has been
found in many forms of cancer cells [
] and might play a
role in the Mosaic Variegated Aneuploidy syndrome. This
raises the possibility of altered expression of BB1
predisposing to chromosomal loss during mitosis and playing a
role in the loss of X chromosome material in TS [
Obviously, it must be recognized that the differential exon
usage of BUB1B may also be a consequence of the 45,X
monosomy itself [
]. Further studies are required to
discern the role of this gene in other sex chromosome
Similarly, through studies on embryonic stem cells
(ESCs), Robertson et al. and Zvetkova et al. have
described a general DNA hypomethylation in XX ESCs
that generate XO cells comparable to XY ESC [
As DNA hypomethylation increases and XO cell
generations are gradually acquired, the X-to-autosome ratio
decreases similar to that observed in control XY cells,
indicating a loss of one of the two X chromosomes
during serial passaging . Thus, this globally reduced
methylation level is associated to X chromosome
instability. Additionally, methylation of differentially
methylated regions (DMR) can be restored, but it is
coincident with complete loss of an X chromosome in
ESCs. Observations indicate that DNA hypomethylation
in XX ESCs is attributable to the presence of two
(active) X chromosomes rather than to the absence of a
Y chromosome [
]. Global DNA hypomethylation and
complete or partial deletion of DNA sequences from
one of the two X chromosomes are associated with
reduced levels of the de novo DNA methyltransferases
Dnmt3a and Dnmt3b. Therefore, it would seem that
global DNA hypomethylation in XX ESC lines occurs
gradually during the embryonic passage of these cell
lines and is probably due to a reduced level of the de
novo DNA methyltransferases Dnmt3a and Dnmt3b.
DNA hypomethylation can be restored in the late phase
of embryonic passage of these ESCs and is coincident
with complete or partial loss of DNA sequences from one
of the two X chromosomes [
]. These findings support
the hypothesis of retention of methylation provides a
selective pressure for deletion of all or part of one X
chromosome. Thus, the selection against loss of methylation
may provide the basis for X chromosome instability.
Given the limited evidence in humans, the findings
described in the XO mouse models can only be
extrapolated in a speculative way to the humans. Unlike their
human counterparts, in which approximately 15% of the
X-linked genes escape X inactivation [
], in the XO
mice, only a few “escapees” [
] have been reported.
This has been used as evidence demonstrating the
mouse X chromosome is depleted of genes escaping X
inactivation, via DNA methylation, which may explain
why the XO mouse has a near normal phenotype [
However, although the DNA methylation pattern on the
X chromosome observed in mice differs from humans,
several similarities have been reported in early embryonic
development in both species. (1) The establishment of
stable XX ES cell lines from both mouse and human
blastocysts is relatively problematic owing to frequent loss
of one of the two X chromosomes [
]. (2) DNA
hypomethylation is globally detected in both murine and
human cell lines [
13, 33, 34, 41, 42
]. (3) This global
hypomethylation can be a causal factor of the instability of the
X chromosome which would condition early loss of the
second sex chromosome in both systems. (4) The
mechanisms of DNA methylation in eutherian mammals have
been evolutionarily conserved. DNA methylation plays an
important role in the regulation of gene expression ,
genomic imprinting [
], and X chromosome inactivation
]. In eutherian mammals, two de novo DNA
methyltransferases, Dnmt3a and Dnmt3b, have been shown to be
essential for early embryonic development [
Human DNMT3A and DNMT3B share 98 and 94% of
their amino acid sequence identity with the mouse
Dnmt3a and Dnmt3b [
], which may indicate that their
expression is regulated in a similar way and that
alterations in their functions could originate a similar
phenotype. For example, studies of Dnmt3b null and
ICF mutant mice have shown that Dnmt3b is essential
for mouse embryonic development and that the ICF
mice exhibit phenotypes that resemble some of the
symptoms of the human ICF syndrome [
]. All of this
evidence suggests that it is feasible that the same events
that occur in the embryonic passage of these cell lines in
the murine model could be similar to those occurring in
the human system.
Meiotic versus post-zygotic origin of 45,X monosomy
Meiosis is the cellular division that underlies haploid
gamete development. To achieve the reduction in diploid
genetic complement needed for gamete formation, one
round of DNA replication (S phase) is followed by two
rounds of chromosome segregation (meiosis I (MI) and II
(MII)). Although the role of the meiotic process is to
generate gametes having half the genetic complement of
the germ cells, during meiosis, genetic recombination
occurs so as to increase genetic diversity. Genetic
recombination involves the pairing and synapsis of homologous
chromosomes, followed by genetic information transfer
(crossover) between regions of homologous sequences of
non-sister chromatids of homologous chromosomes.
Additionally, genetic recombination also plays a mechanical
role in the segregation of homologous chromosomes when
they separate at MI. This chromosomal segregation at MI
is ensured by the presence of physical connection between
homologous chromosomes (chiasmata). It has been
suggested that reduced homologous regions between X and
Y chromosomes in male meiosis is particularly susceptible
to chromosomal nondisjunction [
]. Thus, due to the
heterologous nature of sex chromosomes, it is not surprising
that the nondisjunction rate for sex chromosomes is higher
than that for autosomes in male human meiosis [
sperm typing demonstrates that reduced recombination is
associated with the production of aneuploid 24,XY human
]. Therefore, although autosomal aneuploid
conditions are predominantly maternal in origin [
chromosomal aneuploidies occur frequently because of
nondisjunction in the meiosis male [
]. In support of this
assertion, several studies have shown that two thirds of 45,X
monosomy individuals retain the maternal X chromosome
]. In addition, an increased aneuploidy frequency for
sex chromosomes relative to that of the autosomes in both
human sperm karyotypes [
] and FISH analysis of
] supports this concept.
A characteristic feature of MI is genetic recombination
which is initiated by the formation and repair of induced
double-strand breaks (DSBs). Defects in genetic
recombination disrupt DSB repair and result in checkpoint
activation. In turn, checkpoint activation leads to either
apoptosis or the formation of aneuploid gametes. Thus,
it is critical that germ cells monitor these events and
ensure they occur properly. However, heteromorphic
chromosomes of sexually reproducing organisms
(heterogametic sex) are either partially (e.g., XY in human, other
mammalian, and Drosophila males) or completely
hemizygous (e.g., X0 in a number of insects and worms),
representing a special challenge to the repair of DSBs and,
moreover, in evading checkpoint activation. Consequently,
the underlying meiotic program of sex chromosomes
must be epigenetically modified to promote adequate sex
chromosome segregation during meiosis. Therefore, DSB
repair and checkpoint suppression must occur in the
context of a specialized chromatin structure found only
on the X chromosome of males [
]. Specific silencing
epigenetic marks on sex chromosomes alter interactions
with DSB repair and checkpoint machinery to ensure
accurate transmission of the male genome through
meiosis, so as to achieve suitable sex chromosome
segregation. Thus, during mammalian male meiosis, the
heteromorphic sex chromosomes undergo a silencing
process called Meiotic Sex Chromosome Inactivation
] which results in acquisition of repressive
chromatin and transcriptional silencing. In Caenorhabditis
elegans, MSCI is mediated by MET-2 methyltransferase
deposition of histone H3 lysine 9 di-methylation
(H3K9me2). MSCI is important for shielding the
hemizygous X chromosome linked gene expression from
checkpoint machinery . It is essential for preventing the
expression of a small number of deleterious Y
chromosome-linked genes during male meiosis in mice,
which would result in pachytene arrest and elevated
germline apoptosis [
Thus, alterations in the pathways that mediate DSB
repair on the hemizygous regions of sex chromosomes
in male meiosis could lead to sex chromosome
monosomic gametes. Variations in the recruitment of DSB
processing factors and checkpoint proteins to DSBs in
germ lines with altered chromatin could modify the
specific histone code on DSB repair and checkpoint
silencing. Also, analyses of germline chromatin epigenetic
signatures in Caenorhabditis species indicate that either
H3K9me2 or H3K9me3 can be enriched on sex
chromosomes and mediate transcriptional silencing [
Modifications of these epigenetic marks could lead to checkpoint
activation and consequently to the formation of nullisomic
gametes for sex chromosomes. However, further studies
are necessary so as to understand the increased frequency
of sex chromosome aneuploidy associated with human
meiosis resulting in developmental disorders including
Turner and Klinefelter syndromes.
On the other hand, although chromosomal instability
is a well-known feature in cancer cells, it also is a
prominent trait in early embryonic development. Early
embryonic cell division is distinguished by the suppression
of the cell cycle checkpoints [
], with epigenetic
reprogramming of the genome and total demethylation of DNA
sequences to exclude imprinted loci (totipotent stem
cells). Consequently, chromosomal instability in
blastomeres occurs. Therefore, abnormal post-zygotic epigenetic
reprogramming can cause catastrophic consequences.
Molecular cytogenetic techniques for pre-implantation
genetic diagnosis of individual blastomeres have
demonstrated that 15–85% of early embryos have numerical
chromosomal abnormalities [
]. This chromosomal
instability involves monosomy–trisomy for whole
chromosomes, parental disomies, and complex patterns of
segmental deletions, duplication, and amplification that are
reciprocal in sister blastomeres, implying the occurrence
of breakage–fusion–bridge cycles . These findings
correspond on the one hand with the complex structural
chromosomal aberrations observed in individual with
birth defects [
], and on the other hand, they also explain
the high incidence of aneuploidy embryonic losses (failure
in the implantation process). Thus, monosomy of the sex
chromosomes could be a relatively common event and
explain the high lethality observed in 45,X embryos.
Although meiotic nondisjunction is assumed to be the
principal mechanism in explaining the aneuploidies of
sex chromosomes, several lines of evidence suggest that
45,X monosomy may have a post-zygotic origin in
accordance with the early embryonic chromosomal
instability mentioned above. First, the incidence of 45,X
karyotypes in early embryos is notably higher than that
inferred of nullisomic gametes for a sexual chromosome
]. Second, in contrast to the majority of
constitutional autosome aneuploidies, the 45,X conceptuses that
retained paternal X chromosome are not associated to
maternal age, i.e., are not caused by a maternal meiosis I
error . Thirdly, a very large proportion of 45,X
embryos acquired their 45,X cell line after in vitro
]. Lastly, human embryonic stem cell
lines loose one sex chromosome during in vitro passage
. Thus, the most likely scenario seems to be that of
45,X monosomy arising post-zygotically through a
One hypothesis to explain the survival of the
postzygotic 45,X cell line in contrast to the normal euploid
cell line (46, XX or 46 XY) is that epigenomic
deregulation could promote rapid survival selection of 45,X cells.
Thus, the loss of a second sexual chromosome may
introduce a global epigenetic effect as a cellular response
to overcome the detrimental effects of monosomic cells.
This may indirectly facilitate the process of adaptive cells
by producing a fitness advantage only after a secondary
and required genetic event has occurred [
Phenotypical consequences of the epigenetic modifications in 45,X monosomy
The clinical consequences logic attributed to 45,X are
direct dosage effects of genes on the sexual chromosomes,
i.e., TS is due to complete or partial loss of DNA
sequences in the second sex chromosome which provokes
haploinsufficiency of genes that are normally biallelically
expressed from both sex chromosomes and escape from X
chromosome inactivation. In humans, ~ 15% of X-linked
genes escape X-inactivation [
] so that only a small
number of genes are predicted to contribute to dosage
imbalances in 45,X monosomy. A large effort has gone
into attempting to identify genomic regions on sex
chromosomes that contain the critical genes associated with
the TS phenotype. Cytogenetic characterization of
structurally abnormal X and Y chromosomes has provided
support for the hypothesis of distinct TS loci and a unique
candidate gene probably does not exist. Consequently,
certain clinical features in TS have been mapped to
specific areas of the sex chromosomes: short stature to the
X and Y tip of the short arm and ovarian function to both
the short and long arms, i.e., different clinical features
may be due to different genes. Although several candidate
genes have been proposed to explain what causes the TS
phenotype, the only gene that has been proven to be
associated with clinical features of TS (skeletal anomalies and
short stature) is the SHOX gene (short stature
homeoboxcontaining gene, NM000451) [
]. Therefore, the
prevailing hypothesis of a gene dosage imbalance following
loss of one sex chromosome material is questioned. Thus,
other hypotheses to explain both the clinical features and
the variability of expression in TS have been proposed.
The absence of the second sexual chromosome in TS
has led authors to speculate that there may be genes
present on the X chromosome which are expressed
differently depending upon whether they are maternally
or paternally inherited [
55, 56, 58–60, 79, 84–88
other words, a 45,X TS patient has an increased
likelihood of certain features depending on whether her X
chromosome was inherited from her mother or father. It
seemed based on a number of original studies that
patients with TS that inherited their only X chromosome
from their mother had an increased incidence of
cognitive function disorders, visceral adiposity, and
atherogenic lipid profiles, similar to that of men [
55, 59, 84,
], which led to the hypothesis of X-linked imprinted
genes. However, no human genomic imprinted genes on
the X chromosomes have been identified, and no
significant skewed parental origin effect of the clinical features
of TS patients has been recognized, with the exception
of those exhibiting sexual dimorphism. In addition, other
studies have provided conflicting results in relation to
Xlinked parental effects. Furthermore, the parental origin
of the retained X chromosome may be a confounder
factor of clinically important parameters. Therefore,
from a clinical perspective, a genetic work-up to detect
the parental origin of the remaining X is currently not
indicated in routine care of women with TS .
Alternatively, transcriptome studies have implicated several
genes with altered expression involved in pathways
controlled through epigenetic regulation [
may contribute directly to known pathological
mechanisms identified on prior gene expression profiling in
genes associated to autoimmune diseases; urogenital
congenital malformations; obesity and metabolic
disorders, including type 2 diabetes mellitus (T2DM) and
metabolic syndrome; fetal development and embryonic
lethality; sensorineural hearing loss; and aneuploidies
13, 15, 17
]. The TS phenotype may therefore likely arise
from an abnormal connection of various genetic and
epigenetic factors whose primary source is the
monosomy of the second sex chromosome. On the other
hand, the effects of genetics on epigenetic modeling can
be clustered into those that occur in cis, such as
shortrange effects of SNPs and haplotypes, and those that
occur in trans, including the effects of 45,X monosomy
on chromatin states . In TS, another important class
of trans-acting genetic effects could be due to deleted
second sex chromosome genes which code epigenetic
“reader” and “writer” enzymes. Increasing evidence
suggests that TS features could be caused by altered
regulation and complex interrelations of many genes both on
and outside both sex chromosomes [
13, 14, 16–19
Several studies have compared global gene expression
of 45,X monosomy on peripheral blood mononuclear
], amniotic fluid , human fibroblast cells
] and induced pluripotent human cell lines  to
their euploid and/or 47,XXX trisomic cell counterparts.
Not surprisingly, 45,X cells display a differential global
gene expression pattern separable from euploid and
47,XXX cells. However, it should be noted that the most
differentially expressed genes were not located on the
retained X chromosome. Moreover, thousands of
autosomal genes are differentially expressed between the
sexes in several somatic tissues, with 14% (brain) to 70%
(liver) of active genes affected [
To analyze the functional interpretation of large lists
of genes derived from microarray studies, Database for
Annotation, Visualization and Integrated Discovery
(DAVID) has been employed as an online bioinformatic
resource. By using a novel cDNA based microarray
approach, differential methylation in human X
chromosome in 45,X, 46,XX, and 47,XXX cells has been
]. These studies utilized low-resolution
DNA methylation analyses and have provided evidence
of an altered methylome in 45,X fibroblast cell lines [
], as well as in peripheral blood mononuclear cells
17, 84, 91, 95
]. In a study, DAVID analysis revealed a
differential methylation profile of genes associated with
nuclear chromosome condensation and chromatin
remodeling complex involving histone modification .
When the expression profile of 45,X and 46,XX were
compared, the “skeletal system,” “gonadal development
and function,” “glucose metabolism,” and “epigenetic
regulation” pathways were found to be differentially
expressed in 45,X cells. Interestingly, this methylation
profile correlates with the clinical picture that is displayed by
TS subjects [
]. However, these studies did not examine
a well-characterized cohort of women with 45,X
monosomy. In this context, the leukocyte DNA-methylation
profile was recently investigated when 45,X, 46,XX, and
46,XY subjects were compared by using the
450KIllumina Infinium assay [
]. Genome-wide X
chromosome RNA expression, autosomal DNA-methylation, and
X chromosome methylation profiles clearly discriminated
TS subjects from controls. This study demonstrated
genome-wide hypomethylation, with the most DMRs
showing a medium level of methylation. This
hypomethylation status was extended to repetitive elements.
Interestingly, novel genes including several escape genes, X-Y
homologous gene pairs, and pseudoautosomal genes were
identified. These genes could be related to comorbidities
in TS such as autoimmune diseases, urinary congenital
malformations, premature ovarian failure, and aortic
aneurism formation [
]. Methylation of CpG islands in
gene promoter regions during development, growth, or
disease processes is associated with posttranslational
histone modifications that lead to a locally condensed
inactive chromatin structure and gene silencing.
Genomewide methylation studies have identified this epigenetic
signature in 45,X cells in several tissues, including
leukocytes, skin fibroblasts, buccal cells, liver, and placenta,
adding another layer of complexity to the highly variable
clinical features of TS. Interestingly, X chromosome carries
several genes that are key players in epigenetic regulation.
As mentioned before, an interesting finding of studies
that analyze the differential methylation in 45,X, 46,XX,
and 47,XXX cells is that methylation-based and
expressionbased pathway analyses are complementary rather than
overlapping. These findings could be due to the different
methodologies employed, cell-tissue tested, and specific
epigenetic memory (for example, hypomethylation of
enhancers active during embryonic development, but
dormant in adult tissues [
]). Collectively, the findings
in all of these studies suggest (1) a unique expression and
DNA methylation profile for 45,X cell lines. Consequently,
widespread differences in methylation and changes in
global gene expression distinguish both TS (45,X) and control
(46,XX and 46,XY) subjects. It should be noted that the
most differently expressed genes were not located on the
retained X chromosome but rather on the autosomes. (2)
Sex chromosomal escape genes, X-Y homologous gene
pairs, and pseudoautosomal genes are prone to differential
expression in 45,X subjects. (3) A relationship between
differential gene expression (and increased differential
methylation) and comorbidities exists in TS. This
differential gene expression includes (a) genes located on the
autosomes and X chromosomes; (b) genes associated to
autoimmune diseases; (c) genes associated to a distinct
neuro-cognitive profile; (d) genes associated to urogenital
congenital malformations; (e) genes associated to
reestablishment pluripotency and germ cells (consequently
associated with premature ovarian failure); (f) genes
associated to obesity and disorders in metabolism, including
T2DM and the metabolic syndrome; (g) genes associated to
fetal development and embryonic lethality; (h) genes
associated to sensorineural hearing loss; and (i) genes associated
to aneuploidies. One should also stress that genes involved
in the processes of epigenetic regulation may show a
different DNA-methylation state in 45,X and 46,XX cells
]. This is consistent with the observation mentioned
above of the role the absence of the second sex
chromosome can cause on the deregulation of epigenetic
modifications (Fig. 1).
A striking trait of TS is the wide clinical variability
exhibited by non-mosaic 45,X individuals. However,
intrauterine growth retardation (IUGR) and postnatal short
stature are almost invariable characteristics of the X
chromosome monosomy. These particular features have
been associated with dyslipidemia and T2DM in adult
non-TS subjects. This association is explained by an
epigenetic adaptive mechanism called fetal reprogramming.
Similarly, the IUGR seen in TS fetuses is also related to
dyslipidemia and T2DM in adult TS patients, presenting
with a higher incidence than that of the general
]. Therefore, this epigenetic adaptation could
partially compensate for the haploinsufficiency of the
second sex chromosome in 45,X subjects, who may
exhibit a constellation of clinical findings (in number and
severity) depending on the specific epigenomes of each
45,X individual. Thus, the epigenome of each cell in 45,X
subjects may be the result of haploinsufficiency of the
second sex chromosome genes, and this in turn can have
an influence over the effect of the haploinsufficient
proteins on cells, organs, and tissues. To study this
hypothesis, DNA of leukocytes from a phenotypically
well-characterized cohort of adult patients with 45,X
monosomy and age-matched female controls were tested.
A total of 10,687 DMRs were identified on chromosomes
1, 9, 11, 17, 19 and 22 by linear models adjusting for the
estimated relative cell proportions and age (sex
chromosomes were excluded). To assign biological context,
functional annotation clustering was performed using DAVID,
which allowed for the identification of pathways involved
in the development of congenital heart anomalies and
coronary heart diseases, as well as for enrichment clusters
of blood vessel development and transforming growth
factor β. These pathways may account for the increased
prevalence of congenital heart anomalies and increased
incidence of aortic dissection seen in TS .
Transcription microarray-based studies have found that
mRNA levels of some X chromosome-linked genes parallel
the decrease in gene copy number in 45,X cells. However,
studies of gene expression in 45,X versus 46,XX ESCs found
evidence of under-expression in only a small number of
genes on the X chromosome [
14, 15, 17
]. Conversely, some
other X chromosome-linked genes lacked detectable
decreases in expression in XO compared with euploid cells
]. These differences can in some cases be explained by
gene–gene interactions, when more than one gene in the
same transcriptional pathway is present on the same X
chromosome. Similarly, the fact that some genes on the X
chromosome are not differentially expressed in monosomic
cells can depend on the cell and tissue type. Finally, all gene
expression studies have shown that X chromosome
monosomic cells have many quantitative changes in gene
expression, which is consistent with monosomy of sex
chromosome leading to disturbances of transcriptional
networks. Additionally, apart from simple transcriptional
effects, a hypothesis that may explain TS pathogenesis,
supported by an increasing number of studies that involve
epigenetics, is that the presence of X chromosome
monosomy could act in trans to produce tissue-specific
modifications in DNA methylation and chromatin landscape at loci
distributed throughout the genome [
]. These can then be
inherited mitotically to daughter cells and change and/or
hinder determined modifications in gene expression in
developing tissues (Fig. 1).
Contrast of epigenetic modifications in sex chromosome aneuploidies
Both TS and Klinefelter syndrome are associated with
several clinical features that show a broad range of
interindividual expressivity and whose molecular mechanisms
are poorly understood. Comprehending the effect of
both the loss (45,X) and the gain (47,XXY) of the X
chromosome on gene transcription and its correlation to
their variable clinical phenotypes would be very helpful.
Thus, systematic studies comparing genome-wide
expression as well as epigenetic modifications of sex
chromosome aneuploidies, e.g., 45,X versus 47,XXY, could help us
gain insight into the mechanisms involved in their
pathophysiology. Several studies have been conducted focusing
only on one or another condition. In addition, such
studies have analyzed a limited number of samples from a
single available tissue (schizophrenic brain [
], whole blood [
84, 91, 102, 103
], fibroblast [
or amniotic fluid [
]). To date, this issue has been
addressed in both populations only by Sharma et al. [
This study demonstrated that loss of an X chromosome
has a much more profound consequence than gain of an
additional X chromosome, as about five times more loci
are affected in TS than in KS subjects. In addition, when
only autosomal CpG sites were analyzed, approximately
80% of the modifications in TS individuals represented
hypomethylation of these loci, whereas nearly equal
numbers of hypo- and hypermethylated CpG sites were
observed in KS individuals [
]. Therefore, gene ontology
analysis and the etiopathogenesis in both conditions may
be completely different.
Several general conclusions can be reached by analyzing
the results of the published articles on gene expression
and epigenetic changes in the aneuploidies of sex
chromosomes. First, gene ontology analysis reveals different
clusters of genes involved in the clinical features of both
TS and KS. Second, numerical abnormalities of the sex
chromosomes affect not only the status of methylation in
X-linked loci but also, and mainly, large numbers of
autosomal loci. Third, in samples of TS subjects, the affected
loci are characterized by intermediate methylation levels
in 46,XY males/46,XX females. Nevertheless, samples
from TS subjects are still closer to female than to male
samples. Lastly, samples of KS individuals are significantly
less methylated than samples of 46,XX subjects on
strongly silenced loci on the X chromosome, which
indicates a non-optimal X chromosome inactivation process
in KS individuals. Although, in autosomal loci, samples
from TS individuals are distinctly different from those
exhibited by 46,XX and 46,XY subjects, samples from
subjects with chromosomal constitutions 46,XX, 46,XY,
and 47,XXY display a more or less similar methylation
pattern. However, the second X chromosome inactivation
in KS seems to be less operative than in 46,XX subjects.
This opens the possibility that the specific DNA
methylation-based biomarkers identified in these studies
may largely distinguish samples of TS and KS subjects from
female and male samples, respectively. Furthermore, these
biomarkers can also be used for detailed molecular analysis
of differential patterns of DNA methylation and epigenetic
marks associated with sex chromosome aneuploidies.
Haploinsufficiency and its epigenetic consequences in 45,X monosomy
Although the role of epigenetics in TS is currently far
from understood, an increasing number of studies have
demonstrated the key role of DNA methylation and gene
expression on the etiopathogenesis of TS. As summarized
in Table 1, various decontrolled (under/overexpressed) X
chromosome-linked gene products are epigenetic
modulators, thereby deregulating epigenetic mechanisms in TS.
In turn, these disturbed mechanisms might contribute to
the observed features and associated complications in TS.
O-GlcNAcylation: a pathway contributing to epigenetic actions in 45,X monosomy
A number of studies have exposed several autosomal
genes which are differentially expressed between XX and
XO in human and mice models, revealing transcriptional
changes due to monosomy of the X chromosome. In the
mouse model, the findings obtained by microarray and
qPCR were not fully concordant, illustrating the
difficulty in ascertaining modest fold changes, such as those
expected for genes escaping X chromosome inactivation.
Remarkably, considerable variation was observed between
tissues suggesting that inactivation patterns may be
]. However, transcriptional analyses of
XX, XY, and XO mice have uncovered a number of genes
that exhibit both sex differences in gene expression and
deregulation with X monosomy, such as the OGT gene,
leading to genome-wide disturbances . This gene
encodes O-linked N-acetylglucosamine transferase (OGT),
which adds O-GlcNAc onto serine and threonine residues
of proteins. The importance of O-GlcNAcylation as
critical posttranslational modifications for a wide array of
cellular processes has been previously highlighted [
]. Interestingly, OGT is located on the X chromosome
near the Xist locus (in humans at Xq13.1 and in mice at
XqD). This location suggests that OGT is subject to the
control of dosage compensation mechanisms in females
] so that its refined control is necessary for
normal development and that its deregulation can
contribute to an increased risk for a number of associated
conditions in TS . This hypothesis is supported by its
involvement in disorders including cancer [
], cardiovascular disease [
], and T2DM
]. This role for OGT on health could be due to the
fact that O-GlcNAcylation has been identified on a large
pool of intracellular proteins that have wide-ranging roles,
including several epigenetic actions such as the complex
formation with regulators of DNA demethylation,
transcriptional repressors, transcriptional activator, histone
methyltransferase, and polycomb repressive complex 2
]. Moreover, OGT is able to modify histones
] and to alter the C-terminal domain
(CTD) of RNA polymerase II [
]. The epigenetic role of
O-GlcNAcylation regulate complex processes such as cell
cycle progression, cell signaling, and embryonic
], among others. Thus, OGT has emerged as a
highly regulated nutrient-sensing epigenetic modifier that
could modulate diverse expression networks, and its
potential deregulation may contribute to the variable
clinical features observed in TS patients.
Therefore, an abnormal expression of OGT in
maternal or paternal retained X chromosomes might represent
a link that explains the high lethality observed in 45,X
conceptuses and the early-life origin of adult disease.
Consequently, the increased risk of embryonic lethality
and for common diseases of adult life could be a
consequence of an OGT escape from the X chromosome
inactive in the placenta. However, the parental origin of
the retained single X chromosome does not appear to
influence fetal survival [
], and the factor(s) causing
ENPP1 (ectonucleotide pyrophosphatase/ Related to regulation of carbohydrate metabolic process
TRIB3 (tribbles pseudokinase 3)
Involved in insulin signaling
SLC2A14 (solute carrier family 2 member Glucose transporter (GLUT)
14 (facilitated glucose transporter))
RASD2 (RASD family, member 2)
Interfere with the functional activity of TSHR, FSHR, and LHCGR
RPS4X (ribosomal protein S4, X-linked)
A component of the 40S subunit
An essential eukaryotic translation initiation factor. It is required for the binding No change in gene
of the 43S complex to the 5′ end of capped RNA expression 45,X cells (11)
ZFX (zinc finger protein, X-linked)
A transcriptional regulator for self-renewal of both stem cell types
KDM6A (lysine demethylase 6A)
A tetratricopeptide repeat (TPR) protein. It catalyzes the demethylation of tri/
dimethylated histone H3. Importance for reestablishment of pluripotency and
germ cell development
KDM5C (lysine demethylase 5C)
Involved in the regulation of transcription and chromatin remodeling
USP9X (ubiquitin-specific peptidase
A protein similar to ubiquitin-specific proteases. Mutations may cause changes
in the neuronal migration and axonal growth, resulting in intellectual disability
A gated pore that translocates ADP from the cytoplasm into the
mitochondrial matrix and ATP from the mitochondrial matrix into
the cytoplasm. Permeability transition pore complex (PTPC): regulates
the release of mitochondrial products that induce apoptosis.
Involved in spermatogenesis
Paracrine regulation of follicular development
A serine threonine protein kinase similar to the catalytic subunit of cyclic AMP
dependent protein kinases. Involved in renal epithelial morphogenesis and in
macrophage and granulocyte maturation. Deletion causes sex reversal disorder.
Signal transduction (inferred from biological aspects of ancestor)
SLC25A6 (solute carrier family 25
CLDN11 (claudin 11)
STC1 (stanniocalcin 1)
PRKX (protein kinase, X-linked)
LANCL3 (LanC lantibiotic synthetase
component C-like 3)
ALAS2 (5′-aminolevulinate synthase 2)
GPR34 (G protein-coupled receptor 34)
JPX transcript (XIST activator)
An erythroid-specific mitochondrially located enzyme. It catalyzes the first step
in the heme biosynthetic pathway.
Upregulated (> 2.19-fold)
in 45,X cells (11)
An integral membrane protein. It mediate signals to the interior of the cell via
activation of heterotrimeric G proteins that in turn activate various effector
proteins, ultimately resulting in a physiologic response
A nonprotein-coding RNA transcribed from a gene within the X-inactivation
center. It participates in X chromosome inactivation by activating XIST on the
inactive X chromosome
UBA1 (ubiquitin-like modifier activating It catalyzes the first step in ubiquitin conjugation to mark cellular proteins for
enzyme 1) degradation. It also may function in DNA repair.
A multi-pass membrane protein that is localized to the endoplasmic reticulum
and hydrolyzes several 3-beta-hydroxysteroid sulfates, which serve as metabolic
precursors for estrogens, androgens, and cholesterol. Mutations in this gene are
associated with X-linked ichthyosis (XLI
the great lethality in human 45,X pregnancies do not
seem to be X-imprinted.
Female rodents, who have experienced malnutrition
during perinatal development, have an increased
incidence of dyslipidemia [
]. Patients with TS present
with low birth weight/length [
98, 118, 119
subsequently with an increased risk for weight gain [
metabolic syndrome [
], impaired glucose intolerance
and T2DM [
84, 121, 122
], and hypertension and
ischemic heart disease [
84, 87, 119, 122
]. As O-GlcNAcylation
is involved in the development of T2DM , it would
not be unusual to imagine an involvement of OGT in
the pathogenesis of these disorders in TS. An increased
risk for T2DM among adult patients with TS, especially
those with an isochromosome Xq has been observed
]. A proposed explanation for this increased risk
could be that haploinsufficiency for unknown Xp gene(s)
constitutes a “first hit” that causes the basic deficit in
pancreatic β-cell function seen in 45,X patients. Excess
dosage of Xq genes in isochromosome Xq may provide a
“second hit” that exacerbates the deficit, perhaps by
altering other genes involved in pancreatic β-cell
development and function or survival, and/or by stimulating
lowgrade chronic autoimmunity that injures, but does not
obliterate the β-cells [
Interestingly, the human OGT gene is located on the
Xq13 near the Xist locus, so that TS subjects with
isochromosomes Xq have two OGT alleles on the same X
chromosome, and thus, they could potentially
overexpress OGT. In support of this hypothesis, an increased
risk for T2DM has been observed among patients with
Klinefelter syndrome (47,XXY) and 48,XXYY who have
supernumerary copies of OGT, even in the absence of
]. However, no study has been
published so far in TS patients with an isochromosome Xq
in which OGT levels have been tested and further
research is needed to determine if this could be the
cause for the increased incidence of T2DM in these
patients. Similarly, although all observations suggest a
relationship between OGT and the TS phenotype,
particularly relating to cognitive function, visceral
adiposity, and carbohydrate and lipid profiles, it still
remains unclear whether OGT is deregulated in TS
individuals. Consequently, further examination of OGT
expression in these individuals would be of interest.
Alterations of the immune response in Turner syndrome
Another key player gene on the X chromosome involved
in epigenetic regulation is the UTX (ubiquitously
transcribed tetratricopeptide repeat on X chromosome) gene.
Turner syndrome is also associated with alterations of the
immune response. T cell immune alterations and their
relationship to haploinsufficiency of the UTX gene in the
context of epigenetic regulation have been reported in TS
]. A gene expression microarray analysis
performed on peripheral blood mononuclear cells (PBMCs)
was carried out to identify potential pseudoautosomal
Xlinked genes that contribute to immune alterations in TS
and to determine whether the gene expression in immune
cells was altered in TS subjects. A total of 1169 unique
genes showed differential expression between TS and
control female PBMCs, including 35 genes on the X
chromosome. UTX or KDM6A located at Xp11.3 [
was found to be among the top 10 X chromosome linked
genes with the largest decrease in expression [
Interestingly, UTX is the only gene among these
candidates that escapes X inactivation [
] and is a histone H3
lysine 27 (H3K27) demethylase that epigenetically
regulates gene expression. In addition, mice with T cell-specific
deletion of UTX have an increased H3K27 methylation
and a decreased expression at Il6ra and other Tfh (T
follicular helper)-related genetic loci [
]. T cell-specific
UTX deficiency knockout mice also have impaired
clearance of chronic viral infection due to decreased
frequencies of Tfh cells, which are critical for antibody
generation by B cells. Thus, UTX is required for optimal
CD4+ T cell differentiation to Tfh cells during chronic,
but not acute, viral infection [
]. In parallel, reduced
numbers of circulating CD4 CXCR5+ T cells (a measurable
substitute of antibody production from Tfh cells) have
been demonstrated in TS subjects with a decreased UTX
expression in immune cells [
] compared to female
controls. Thus, decreased UTX expression in TS subjects
might increase their predisposition to viral infections due
to Tfh cell deficiency with subsequent reduced antibody
levels. Although all of this data suggests that UTX
haploinsufficiency in TS individual immune cells has functional
consequences, it is unclear whether TS patients also have
an increased predisposition to chronic infection, for
example, chronic otitis media, due to decreased Tfh cell
numbers. In any case, the findings of decreased expression
of UTX in TS immune cells, as well as the mechanism by
which UTX affects CD4+ T cell differentiation to Tfh cells,
are important steps toward understanding immune
alterations in TS.
There is a well-described increase in intrauterine lethality
in 45,X conceptuses [
]. The formation of 45,X embryos
occurs very commonly during human reproduction, but
subsequently, 99% of these embryos are spontaneously
]. Thus, 45,X is considered to be the
most frequent genetic abnormality in the human species
as it is present in approximately 2% of all conceptions that
survive long enough to be clinically recognized
pregnancies and about 15% of spontaneous abortions are 45,X.
Therefore, early lethality is the most prevalent phenotype
of the 45,X karyotype. In contrast to humans, XO mice
are anatomically normal, fertile, and viable with no
prenatal lethality . Consequently, human TS cells or
mouse models cannot be used to study the causes of early
lethality in 45,X embryos. An alternative model to study
the embryonic lethality is human XX ES cell lines, as these
cells can differentiate in vitro into cells from the three
embryonic germ layers, as well as into extra-embryonic
cells. As mentioned above, a well-known feature of XX ES
cell lines is the frequent loss of one of the two X
33, 34, 40
The high lethality of 45,X conceptuses occurs especially
during the implantation period [
], which suggests the
involvement of rather general etiopathogenic mechanisms
]. If the 45,X embryos break through a certain barrier,
then these global etiopathogenic mechanisms would
provoke several accumulated related fetal or congenital
defects. Three non-excluding hypothesis can be postulated
to explain the high frequency of early lethality in 45,X
embryos. First, in both morula and blastocyst stages, the
pluripotent 45,X cells could be unstable, and therefore,
differentiation is triggered too quickly and/or randomly.
In this scenario, one would expect that the
reprogramming of 45,X cells to iPSCs is not able to occur, since
pluripotent 45,X cells would automatically degenerate or
not transform into ESCs. However, using either skin
fibroblasts or amniocytes from 45,X subjects, 45,X iPSCs have
been generated [
], able to stably maintain their
chromosomal constitution and with phenotypes similar to ESCs.
In addition, these 45,X iPSCs were used for further assays
including global gene expression analysis and
tissuespecific directed differentiation [
The second hypothesis postulates that 45,X ESCs
undergo a differentiation blockade, and consequently,
certain tissue and/or structures cannot be formed properly
leading to embryonic loss. In this case, 45,X embryos
would either not truly differentiate into one or more of
the three germ cell layers (ectoderm, endoderm, or
mesoderm) or tolerate specific defects in certain cell
lineages (e.g., cardiac mesoderm). However, 45,X iPSCs
become rather complex teratomas and could produce,
upon directed differentiation in vitro, specific lineages
morphologically and functionally indistinguishable from
those produced from ESCs or euploid iPSCs.
Thus, although the existence of subtle lineage-specific
differentiation defects causing multi-organ failure and
embryonic loss in TS fetuses is possible, a third scenario
seems to be more feasible: inadequate global
placentation and/or unbalanced cell proliferation/differentiation
may disturb coordinated embryonic and extraembryonic
tissue growth, eventually causing the embryos death.
The findings of a reduced gene expression in X
chromosome-linked genes in 45,X iPSCs support the
existence of a different and more general etiopathogenic
A differential expression profile of genes that escape X
inactivation was analyzed in a microarray analysis
(Affymetyrix) from human XX ES cell lines [
RNA was extracted from populations of undifferentiated,
in vitro and in vivo differentiated cells derived from
human normal and XO ES cell lines. Out of 37 analyzed
genes that escape X inactivation, 21 genes were
expressed in differentiated and undifferentiated human
XO ES cell lines. From these 21 identified genes, only
three ARSE, STS, and TBL1X fulfilled the criteria of
being a pseudoautosomal gene and to be expressed in
46,XX cells, but not expressed (or expressed at a very
low level) in 45,X ES cells. None of these three candidate
genes showed monoallelic expression, which implies that
genomic imprinting does not play a major role in the
early lethality of 45,X embryos. Gene expression
microarray data was divided into lists of genes enriched
in different tissues, and their gene expression of 46,XX
and 45,X were compared in differentiated ES cell lines.
The only tissue where many genes were expressed at
higher levels in XX cells as compared to XO cells, both
in vivo and after in vitro differentiation, was the
placenta. All placental genes were expressed at least 5-fold
higher in XX ES cells as compared to 45,X ES cell lines.
Of these genes, STS and CSF2RA are X chromosome
linked genes that escape X chromosome inactivation and
were found to be highly enriched in the placenta.
However, the STS gene has no active homolog on the Y
], and therefore, it is not likely that
haploinsufficiency of this gene causes the abnormal
placental differentiation. CSF2RA (colony-stimulating factor
2 receptor alpha) gene encodes the alpha subunit of the
receptor of granulocyte-macrophage colony-stimulating
factor (CSF2 or GMCSF) which is an essential autosomal
gene for normal placental development [
]. It is
remarkable that CSF2RA is expressed 9.5-fold more in
46,XX cells than in 45,X cells [
]. It is therefore
possible that haploinsufficiency of CSF2RA may lead to
abnormal placental differentiation. This, in turn, may
cause an epigenetical downregulation of many placental
genes, including CSF2RA itself, which might explain its
higher differential expression level in 46,XX than in 45,X
ES cell lines. It is also intriguing to see that Csf2ra in
mice is an autosomal gene. This might explain the less
severe phenotype seen in XO mice versus that seen in
45,X embryos, a hypothesis previously explored [
This also suggests that the genes involved in 45,X early
lethality phenotype are regulated epigenetically by a
pseudoautosomal X chromosome linked gene. Overall,
these data point out that at least one of the reasons for
the early lethality of 45,X embryos is abnormal placental
differentiation as a result of haploinsufficiency of
Xlinked pseudoautosomal genes. The effect of X
monosomy on placental differentiation may occur very early
on and may therefore have a general influence on
various components of the placenta.
In addition, 45,X iPSCs have been also used for studies
of global gene expression analysis and tissue-specific
directed differentiation [
]. Lower levels of gene
expression were found for the pseudoautosomal gene PPP2R3B
in multiple clones when compared with euploid cell
controls. These 45,X cell clones could be transformed
into neural-like, hepatocyte-like, and heart-like cells.
PPP2R3B is a pseudoautosomal gene whose product is a
subunit of protein phosphatase 2A which acts as a
negative regulator of cell proliferation and is important for
the correct exit of mitosis during early embryonic cell
]. Tentatively, the haploinsufficiency of
the PPP2R3B gene in 45,X conceptuses might alter cell
proliferation during early embryonic development and
indirectly also affect tissue-specific differentiation due to
improper synchronization. Although, no increased
proliferation in 45,X iPSCs when compared with the
controls was shown , it is possible that the mitotic
defect is more relevant in vivo and/or becomes apparent
when 45,X blastocysts differentiate. In addition, these
clones displayed insufficient upregulation of CSF2RA
during embryoid body formation [
]. Taken together,
the findings described in 45,X iPSCs support the notion
that abnormal organogenesis and early lethality in 45,X
conceptuses are not caused by a tissue-specific
differentiation blockade but may rather involve other
abnormalities including impaired placentation.
On the other hand, the essential role of OGT for
embryonic and extraembryonic tissue development has
been demonstrated in the knockout model. Ogt
knockout mouse embryos die in the blastocyst period [
suggesting that OGT is required during pre-implantation
development. Ogt has been identified as having decreased
expression in XO mouse placenta [
] and has not been
shown to undergo genomic imprinting on the inactivated
X chromosome in XX extra-embryonic tissue [
Consequently, Ogt expression is biallelic in XX mouse
trophobast stem cells, as XX placentas have higher levels of
gene expression of Ogt and higher concentrations of
OGlcNAcylated proteins than XY placentas [
the embryonic lethality observed in mice that inherited a
maternal mutant X results from decreased Ogt expression
during pre-implantation development, rather than from
decreased expression in extraembryonic tissues [
However, taking into consideration the fetal reprogramming
hypothesis, the differential dosage of O-GlcNAcylation, and
by extension OGT differential expression between 45,X and
46,XX placentas, may play a role in processes that contribute
to parent-of-origin effects such as gluconeogenesis,
lipogenesis, and cardiac metabolism [
Future implication of epigenetic therapies on
clinical features and associated complications in
Due to the probable impact of epigenetic changes on the
TS phenotype, resulting from X chromosome
monosomy, the potential role of epigenetic treatment for the
care and management of TS should be considered. First,
hypomethylating agents such as 5-azacytidine, while
useful as anticancer epidrugs, are mutagenic and,
therefore, not appropriate for use in other clinical conditions
such as TS [
]. Treatment approaches based on
dietary management can also affect DNA methylation
patterns, and they are known to be nontoxic and safe
and may, thus, be used as potential therapeutic agents in
preventing or improving the associated metabolic
complications frequently seen in adult patients with TS.
The classical example of a methyl donor is the
supplementation with folic acid and vitamin B12. Although not
expected to reduce methylation on hypermethylated loci,
it might prevent losses of methylation at hypomethylated
loci, therefore helping to maintain methylation levels
genome-wide. However, to our knowledge, no studies
have so far examined this type of therapy in individuals
with TS in controlled trials. This therapeutic approach,
based on dietary manipulation, has been demonstrated
to have variable and partly promising effects on
measures of overall health in other disorders . On
the other hand, identification of target master regulator
genes that can play key roles in the development of
clinical features and associated complications, such as
immune deregulation and short stature, may provide
clues to future therapies, both in TS and in other
conditions. Lastly, the most promising of the therapy-based
modifications of gene expression is the technique of
epigenetic editing in which specific epigenetic enzymes,
such as a DNMT or HAT, are recruited to specific genes
by means of a lab-engineered DNA-binding domain.
Consequently, epigenetic changes are actively
superscript, leading to potentially continuing modulation of
gene expression. Plausibly, the expression of
underexpressed X chromosome-linked genes might produce
physiological levels that might improve short stature and
impaired glucose and lipid metabolism, among other
complications of TS.
Summary and future perspectives
Even though the etiology of TS (monosomy of the
second sex chromosome) has been known for many
decades, how this aneuploidy leads to the TS phenotype
is still not well understood. Clinicians and scientists
believed that TS was a contiguous gene syndrome resulting
from deleted genes carried on the second sex chromosome
which gave rise to short stature, gonadal dysgenesis, typical
visible dismorphic stigmata, and associated urinary,
cardiovascular, skeletal, and endocrine abnormalities. So far, the
prevailing hypothesis, based on rare structurally abnormal
X and Y chromosome case reports, supports gene dosage
effects, in which decreased expression of X
chromosomelinked genes lead to direct altered biological outcomes and
interrupts and/or modifies many downstream
transcriptional pathways. Thus, it is widely assumed that the TS
phenotype results from dosage imbalance of the genes
located on the second sex chromosome (X or Y
chromosome). Although the deletion of genes on the second sex
chromosome would theoretically lead to a 0.5-fold
decrease in the gene expression level, the mRNA levels of
many X chromosome-linked genes deviate from this.
Whereas most researchers have tried to identify these
genes and its contribution to TS features, the underlying
cause of this gene expression variation has been largely
ignored. In this respect, epigenetic mechanisms are of
crucial importance in gene expression and thus might play
a central role in the development of the TS phenotype.
DNA methylation and mRNA transcription studies suggest
a disruption of pathways involving the “skeletal system,”
“gonadal development and function,” “glucose metabolism,”
and “epigenetic regulation” and that various genetic
mechanisms may contribute to the clinical features of TS and its
Studies on the expression of mRNA in cells and tissues
with X chromosome monosomy have shown that, while
some genes on the X chromosome are under-expressed,
other subsets of genes on other chromosomes show an
altered expression. This could be due to a variety of
mechanisms including the activity of transcription
factors encoded on the X chromosome or elsewhere in
the genome that are affected by this aneuploidy, leading
to alterations in DNA methylation, posttranslational
histone modifications, nucleosomal core assembly, and
chromatin remodeling through miRNAs and lncRNAs.
Recent results support the synthetic view to explain the
etiopathogenesis of TS. The analyses of the epigenome
of TS subjects have demonstrated DMRs that, although
enriched on the X chromosome, are also involved in
most of the chromosomes. Significantly, several of these
DMRs were functionally correlated with pathways
pathologically linked to developmental defects in TS.
Surprisingly, epigenetic mechanisms have been poorly
evaluated in TS. Most TS studies have focused on
genetic aspects, ignoring the current evidence that
points toward the contribution of epigenetics to the TS
phenotype. Therefore, in this review, we have tried to
summarize and to assess the limited information available
on the effect of epigenetics in the etiopathogenesis of TS.
We have also offered further evidence for the role of
epigenetics on short stature, embryonic lethality, cardiac
defects, impaired glucose, and lipid metabolism that have
been extracted from methylation and gene expression
studies, including those of TS patients. As TS probably
results from gene expression disturbances, research of the
molecular mechanisms that regulate phenotypic
expression require an understanding of the transcriptome
differences present in 45,X monosomy cells and tissues, which
have been previously explored by several studies. However,
the extensive natural gene expression variation occurring
in both 45,X and 46,XX cells complicates the
identification of changes related to 45,X monosomy per se. In this
review, we proposed that the under-expression of several
genes modifies the chromatin landscape of the nuclear
compartment in 45,X monosomy cells. These
modifications would lead to a general disturbance of the
transcriptome that might explain some of the clinical features seen
in TS. Since constitutive global hypomethylation and
specific hypermethylated loci in TS affect multiple tissues,
including blood, it may provide useful biomarkers for TS,
as well as pharmacological targets for therapeutic
intervention. Significantly, recent epigenetic therapies
(epidrugs and epigenetic editing) are already being used
for cancer and epilepsy and might offer new, different, and
innovative possibilities for the treatment of short stature
and for the common adult complications seen in TS
patients. To our knowledge, no studies have so far
explored epigenetic therapy in TS. However, it may
provide potentially important novel pathways in the care
and management of TS patients.
An interesting focus for future research may be the
epigenetic involvement of the GH-IGF-I axis in the
etiopathogenesis of short stature, hearing loss, and common
diseases of adult life in TS. A hypothesis proposed in
1990 by Barker is that IUGR has a causal relationship in
the origins of hypertension, coronary heart disease, and
DMT2 in adulthood, so that a stimulus or insult at a
critical, sensitive period of early life has permanent
effects on structure, physiology, and metabolism later in
life (fetal programming) [
]. Fetal programming
may result from adaptations that occur when the maternal–
placental nutrient supply fails to match the fetal nutrient
demand. Intrauterine growth retardation is a constant
clinical feature of X chromosome monosomy [
Multiple factors can condition the presence of IUGR in TS
]. In general, IUGR decreases serum insulin growth
factor-1 (IGF-1) levels, and a reduced mRNA IGF1
expression has been demonstrated in TS fibroblasts, which
suggests a reduced autocrine/paracrine action of IGF-1 in TS
]. Additionally, reduced free IGF-I and increased
IGFBP-3 proteolysis in TS, modulated by female sex
steroids, has been described in women with TS [
is an epigenetically regulated gene that has two promoters,
alternative exon 5 splicing, and multiple termination sites
]. Conservation nucleotides and amino acids of
IGF-I in human and rat species, as well as a comparable
expression of multiple mRNA variants, have been noted for
both species . Also, a profile of epigenetic signatures in
the GH-IGF-1 axis has been described in IUGR model rats
(a recent review examined this topic [
]). Due to the
evolutionarily conserved and biochemical and genetic
similarities of the GH-IGF-I axis in both species, this
pattern of epigenetic marks in the murine models might be
extrapolated to the human. The early developmental
epigenetic maturation pattern may be essential for the
maintenance of an optimal GH-IGF-I axis during infancy,
childhood, adolescence, and adult life in TS, and it can be
altered by IUGR. Also, mRNA IGF1 variants might be used
as markers of altered transcriptional regulation and may
function to monitor the growth and response to rhGH in
TS subjects. In addition, a study has shown that hearing loss
in TS was related to serum concentrations of IGF-1 and
]. Also, altered lipid and glucose profiles were
related with a low birth weight and length in TS [
IUGR, and its impact on epigenetic signatures during fetal
life, may play a role in the onset, course, associated
complications, and therapy of common diseases of adult life in this
group of patients. Therefore, TS might be considered
another example of fetal programming and the
consequences of it. For example, although epigenetic mechanisms
can affect the regulation of several gene pathways
throughout life and explain the gene–environment interaction,
more importantly, they are potentially modifiable and
consequently reversible. Growth hormone (rhGH) therapy
at supraphysiological doses is known to improve the final
height of TS subjects. Knowing the regulatory epigenetic
pattern of the human IGF1 gene would help identify an
accessible chromatin around this locus in TS patients. This
may lead to the use of a lower dose of rhGH in TS subjects
with fewer adverse side effects, opening the possibility of a
more personalized medicine.
Another interesting and remarkable mechanism, not
explored until now, but which might gain acceptance in
the next years, it is our understanding of the epigenetic
regulation of transcriptional control of metabolic
diseases of adult life in TS. Metabolic syndrome (MetS)
is a cluster of common disorders that include visceral
adiposity, hypertension, insulin resistance, and dyslipidemia.
This disorder is associated with an increased risk for
cardiovascular disease, myocardial infarction, stroke, and T2DM.
The frequency of each component of MetS in adult TS
patients is high. The atherosclerotic process starts early [
90, 121, 150, 151
] in TS, and epidemiological data suggests
that there is an associated 3-fold increase in the risk of
mortality from cardiovascular and cerebrovascular diseases,
when compared to that of the general female population
]. Thus, the life expectancy in TS is reduced by at least
10 years [
]. An increased risk of insulin resistance,
hypertension, glucose intolerance, dyslipidemia, and liver
dysfunction have been associated with adult TS patients
84, 90, 150, 151
]. Insulin resistance and dyslipidemia are
probably the most common metabolic abnormalities in
normal-weight adult patients with TS. Insulin resistance
and dyslipidemia are frequently related to the disorders in
apolipoproteins, receptors, enzymes, or cofactors in
proteins related to glucose and/or lipoprotein metabolism
]. Peroxisome proliferator-activated receptors (PPARs)
are members of the second class of nuclear receptors [
PPARs act through genomic, as well as nongenomic
mechanisms and their activity can be modified posttranslationally
] and regulated by epigenetic mechanisms. Among
them, PPARγ is involved in adipocyte differentiation, lipid
and glucose metabolism, mitochondrial biogenesis, and
regulation of inflammatory pathways [
]. Exploring the
role of epigenetic regulation, as a potential modifier of the
metabolic phenotype seen in TS, may be of interest in
future research. In particular, analyzing the epigenetic
aspects of genes such as peroxisome proliferative-activated
receptor c coactivator 1 alpha (PPARGC1A) might open
new routes of research, due to altered signaling of this gene
which have been described as contributing to glucose
intolerance, insulin resistance and T2DM and control of
Thus, there is now a growing consciousness in the
clinical field that having the correct pattern of epigenetic
signatures is critical for the development of a normal
phenotype. If epigenetic mark patterns in the GH-IGF1
axis are not properly established or maintained during
the fetal period, as may occur in TS, conditions as
diverse as short stature, hearing loss, hypertension,
T2DM, dyslipidemia, and cardiovascular disease may
appear. However, much more research will be required
before translating these findings into clinical practice.
In conclusion, the phenotypic consequences of epigenetic
modifications in TS are being evaluated. These studies will
require assessment of the epigenetic roles of upstream
effector genes on the X chromosome, including DNA
methylation- and histone modification-pathway genes,
transcriptional genes and non-codifying RNA (miRNA),
and the groups of downstream target genes that are
affected epigenetically by the X chromosome monosomy.
APC/C: Anaphase-promoting complex/cyclosome; CSF2RA:
Colonystimulating factor 2 receptor alpha gene; DAVID: Database for Annotation,
Visualization and Integrated Discovery; DMRs: Differentially methylated
regions; DNMT: DNA methyltransferase; DNMT3B: DNA methyltransferase 3b
gene; DSBs: Double-strand breaks; ESCs: Embryonic stem cells;
H3K27: Methylation of lysine 27 residues at histone 3; H3K9me2: Histone H3
lysine 9 di-methylation; HAT: Histone acetyltransferase;
ICF: Immunodeficiency, Centromeric instability and Facial anomalies;
IUGR: Intrauterine growth retardation; lncRNAs: Noncoding large RNA
molecules; MI, MII: Meiosis I and II, respectively; miRNA: Noncoding
microRNA molecules; mRNA: Messenger RNA; MSCI: Meiotic sex
chromosome inactivation; OGT: O-linked N-acetylglucosamine transferase;
PBMCs: Peripheral blood mononuclear cells; SHOX: Short stature
homeoboxcontaining gene; T2DM: Type 2 diabetes mellitus; Tfh: T follicular helper;
TS: Turner syndrome; UTX: Ubiquitously transcribed tetratricopeptide repeat
on X chromosome gene
This study was supported by a grant from Academie de Recherche et
D’Enseignement Superieur of Belgique (2016-157E).
Availability of data and materials
The PUBMED database of biomedical and life science journal citations
(https://www.ncbi.nlm.nih.gov/pubmed/) was queried using the following
search term: epigenetics AND “Turner syndrome” or methylation AND
“Turner syndrome” or transcriptome AND “Turner syndrome.” The search
returned a total of 64 citations in English between 1977 and 2017. Of the 64
citations, 62 had an abstract available for review. All available unique
abstracts with ≥ 1 citation, a total of 62 abstracts, were reviewed by FAN.
FAN was a major contributor in writing the manuscript. He has made
substantial contributions to the conception and design and analysis and
interpretation of the data. RL has been involved in drafting the manuscript
and revising it critically for important intellectual content. Both authors have
agreed to be accountable for all aspects of the work in ensuring that
questions related to the accuracy or integrity of any part of the work are
appropriately investigated and resolved. All two authors have given final
approval of the version to be published and have participated sufficiently in
the work to take public responsibility for appropriate portions of the content.
Ethics approval and consent to participate
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The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
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