Download Specialist Review Epigenetic variation: amount, causes, and

Specialist Review
Epigenetic variation: amount,
causes, and consequences
Elena de la Casa-Esperón
University of Texas at Arlington, Arlington, TX, US
Carmen Sapienza
Temple University, Philadelphia, PA, US
1. Introduction
The diversity of human phenotypes that we observe is the result of genetic
and epigenetic variation and the interaction of these “biological” variables
with environmental factors. Both large-scale and small-scale genome sequencing
projects, as well as more recent efforts to define structural variation (copy number
variation and subkaryotypic insertions, deletions and rearrangements), have resulted
in an important initial description of the amount and type of genetic variation in the
human genome. On the other hand, the scale of epigenetic variation in the human
population is only beginning to be investigated.
Epigenetic variation may arise by diverse mechanisms but, at the molecular
level, it reflects differences in the spatial configuration of chromatin and its
interactions and function. Multiple biochemical processes (DNA methylation,
histone methylation, acetylation, phosphorylation, sumoylation, etc.) are associated
with these differences. One important consequence of this variability is the resultant
variation in gene expression, although many other effects have also been described
(see the following text).
In the same way that somatic mutations can be transmitted through successive
cell divisions, epigenetic marks can change during the lifespan of an organism
and also be transmitted somatically through subsequent cell divisions. In fact, the
normal phenotypic diversity found between the different cell types of an organism
is, with a few notable exceptions in the immune system, epigenetically controlled.
Interestingly, traits that result from particular patterns of epigenetic modification
can also be transmitted between generations in some circumstances. The term
“epialleles” has been coined to describe such different epigenetic states (Article 36,
Variable expressivity and epigenetics, Volume 1). However, unlike DNA
sequence changes, epigenetic modifications are often reversible at much higher
frequencies than the mutation rate. This is an important characteristic, because
epigenetic marks can be reset between generations and they can change in response
2 Epigenetics
to the environment. Because epigenetic variation can also be genetically controlled,
it constitutes a potentially important link between environmental and genetic
factors (Cui et al ., 1998; Nakagawa et al ., 2001; Sandovici et al ., 2003). Such
a response to the environment could be mediated by metabolic changes that result
in epigenetic modifications (Paldi, 2003; Waterland and Jirtle, 2004; Wolff et al .,
1998). Consequently, epigenetic variability is not only a source of phenotypic
plasticity in response to the environment, but these epigenetic alterations can also,
potentially, be transmitted between generations, with very important implications
in evolution (Rutherford and Henikoff, 2003; Sollars et al ., 2003).
To better understand the relevance of epigenetic variation, we will discuss the
extent (how much?), the origin (what are the causes?), and the implications (what
are the consequences?) of this important source of phenotypic variation.
2. Epigenetic variation: how much?
2.1. Epigenetic variation arises from multiple mechanisms
It is difficult to estimate the precise extent of epigenetic variation because it occurs
at multiple levels and as a result of multiple processes. The epigenetic variation
resulting from inactivation of X chromosome provides a classic example of how
multiple and distinct processes can give rise to very large fluctuation in phenotype
among genetically similar or identical (Fraga et al ., 2005) individuals. In human
females (as in other female mammals), one of the two X chromosomes is inactivated by epigenetic means. Once one of the two X chromosomes is chosen for
inactivation early in development, and the same X chromosome remains inactive
in all descendants of that cell (Article 41, Initiation of X-chromosome inactivation, Volume 1). The inactive X chromosome becomes a cytologically visible
heterochromatic body. This cytological manifestation of femaleness (the Barr body)
is to a large extent (but not completely (Disteche, 1995)) transcriptionally inert.
This means that each single cell expresses only one allele of most (approximately
85%) (Carrel and Willard, 2005) X-linked genes. If both X chromosomes have the
same probability of being inactivated, the “average” women will have the paternal
X chromosome inactive in 50% of her cells and the maternal X inactive in the
remaining 50% of her cells. However, because the process of choosing the X chromosome for inactivation has a large stochastic component (Article 41, Initiation
of X-chromosome inactivation, Volume 1), individual women will have different
patterns of X-inactivation (Figure 1a). In fact, there is a minor fraction of females
in whom >90% of the cells have the same X chromosome inactivated (Figure
1a). These females will have highly preferential expression of either maternal or
paternal alleles of all X-linked genes affected by the inactivation process.
In addition to this partly stochastic, partly genetic variability in the fraction
of cells in which a particular X chromosome remains active (see next section),
there is also population-level and intraindividual variability in the extent of
X-inactivation. It has been documented that a fraction of X-linked genes have
escaped inactivation’ (reviewed in Disteche, 1995). Interestingly, some genes are
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Stochastic
Environmental
IX-inactivation time 1 X-inactivation time 2I /10
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Percentage of cells with active Xce −carryingX-chromosomes
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Figure 1 Origin of X-inactivation variation. (a) Much of the variation in the human results from
the stochastic component of the X-inactivation choice process. Y-axis represents the fraction of women
with the indicated percent of cells with the same X chromosome active (Naumova et al ., 1996 and our
unpublished data). Approximately one-third of women have either X chromosomes inactivated in one-half
of their of cells (purple bar) and approximately 60% of women (purple bar plus green bar) have
X-inactivation ratios between 50:50 and 70:30. However, approximately 7% of women have highly skewed patterns of X-inactivation, that is, greater than 90:10 (blue bar) in favor of the inactivation of a particular X chromosome.
(b) Moving average of change in X-inactivation score in individual females (over nearly two decades; see Sandovici
et al ., 2004) as a function of age. Females who were greater than 60 years of age when the first sample was taken
show significantly more variation over time than younger females. (c) Heritable effects on X-chromosome inactivation
variation. Distribution of X-inactivation ratios in heterozygous Xcea /Xcec mouse females – each circle represents an
individual female mouse (de la Casa-Esperon et al., 2002). The X-controlling element locus affects the probability that
an X chromosome will become inactive, so that X chromosomes carrying the Xcea allele have a higher probability
of being inactivated than X chromosomes carrying the Xcec allele. The observed mean X-inactivation ratio of this
population of females is 25% of cells with an active Xcea -carrying X chromosome
inactivated in some human samples, but escape inactivation in others (Carrel and
Willard, 2005). In addition, the level of expression of such “escapees” also differs
between samples (Carrel and Willard, 2005). Therefore, even genetically identical
women (monozygotic twins) can differ in their mosaic pattern of X-inactivation, the
number of genes that escape X-inactivation, and the levels of expression of some
X-linked genes. In addition, some genes that have been inactivated may become
reactivated as a function of age (Wareham et al ., 1987) or other environmental
factors, although not all X-linked genes appear equally susceptible to reactivation
(Migeon et al ., 1988; Pagani et al ., 1990).
A similar variability in a long-term inactivation phenomenon has been observed
for another class of monoallelically expressed genes located in the autosomes,
the imprinted genes. Imprinted genes are expected to be expressed exclusively, or
4 Epigenetics
nearly exclusively, from the paternal or the maternal copy (Article 37, Evolution
of genomic imprinting in mammals, Volume 1). Several studies have shown that
some imprinted genes (e.g., IGF2 , HTR2A genes) are expressed from both alleles in a small fraction of normal individuals (Bunzel et al ., 1998; Sakatani et al .,
2001), while others (IGF2R) exhibit the reciprocal characteristic of being imprinted
in only a small fraction of individuals (Xu et al ., 1993).
Expression levels between alleles have been found to be variable for several
imprinted genes in human tissues (Dao et al ., 1998; McMinn et al ., 2006), and
have also been observed at nonimprinted autosomal genes. In fact, large-scale transcription profiling studies in humans have shown differential expression of alleles
at a large proportion of loci (up to 54%, depending on the cutoff level of differential expression selected (Lo et al ., 2003)) and, interestingly, the degree of
difference in expression between particular alleles varies between individuals (Lin
et al ., 2005; Lo et al ., 2003; Pant et al ., 2006; Pastinen et al ., 2004). Moreover,
skewing of allelic expression is not necessarily in the same direction: in some
individuals who are heterozygous for the same alleles, the allele that is preferentially expressed differs (Lo et al ., 2003; Pastinen et al ., 2004). This observation
suggests that trans-modifiers and epigenetic variation are involved in the control
of allelic differences in expression, in addition to polymorphisms in cis-regulatory
sequences. Such extensive variation in allelic expression must have a large impact
in generating phenotypic diversity.
2.2. Variability in the biochemical “marks” associated with
epigenetic variation
The types of epigenetic marks that result in allelic variation in gene expression
can be of diverse nature. The best known and most extensively investigated are
covalent modifications of DNA and core histones. DNA methylation at CpG sites
shows a degree of variability between different individuals at multiple loci. This is
the case for imprinted genes like IGF2/H19 and IGF2 R, for which interindividual
variation in methylation patterns has been observed in the differentially methylated
regions associated with their expression (Sandovici et al ., 2003). Interestingly,
alterations in normal methylation patterns of these regions have been associated
with loss of imprinting (LOI), a common observation in several types of cancer (Cui
et al ., 1998; Nakagawa et al ., 2001). Another interesting example of interindividual
variation at an imprinted gene is PEG1 : this gene codes two isoform, one imprinted
(isoform 1) and one expressed biallelically in multiple tissues (isoform 2). However,
in a large subset of human placentae, isoform 2 allelic expression differences are
observed, as well as interindividual variation in methylation of an associated CpG
island (McMinn et al ., 2006).
Interindividual variability in methylation patterns has been also described outside
of imprinted genes or even protein coding regions: this is the case of methylation
differences between humans that is observed in specific Alu repeated sequences
(Sandovici et al ., 2005). These observations reflect the fact that DNA methylation
may have roles in addition to transcriptional control (de la Casa-Esperon and
Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000; Sandovici et al ., 2005).
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Studies in other organisms also support the idea that variation in DNA methylation
could be a widespread phenomenon. For instance, variation in cytosine methylation
has been described in rRNA genes of natural accessions of the flowering plant
Arabidopsis thaliana (Riddle and Richards, 2002), as well as in retrotransposons
(Rangwala et al ., 2006). Also, differentially methylated P1 pigment gene alleles
have been observed in maize (Das and Messing, 1994). Importantly, studies in
Arabidopsis have also shown that both natural and induced methylation changes
can be transmitted to the offspring and result in developmental abnormalities
in some instances (Kakutani et al ., 1999; Rangwala et al ., 2006; Riddle and
Richards, 2005).
3. Epigenetic variation: what are the causes?
Epigenetic variation is the result of three types of processes: stochastic, environmental, and heritable. Variation in X-inactivation illustrates all three of these
processes: during embryogenesis, one of the two X chromosomes is inactivated
in each cell and clonally transmitted through successive mitotic divisions. Because
this choice has a stochastic component (although some deterministic models are also
capable of explaining the observations (Williams and Wu, 2004)), the X-inactivation
patterns of a population of females approximates a normal distribution. The average
female has about half of her cells with the maternal X chromosome inactive and half
with the paternal X chromosome inactive. However, a small proportion of females
show skewed patterns with a particular X chromosome being inactive in most cells
(Figure 1a). Therefore, females are a mosaic for the expression of X-linked genes,
and not even genetically identical females need show the same mosaic pattern.
The so-called skewing of X-inactivation is not always the rare consequence
of the stochastic nature of the choice process. In some instances, skewing is
the result of selection against X chromosomes carrying deleterious mutations,
and the cell type-specificity of this skewing, as in X-linked agammaglobulinemia
(skewing for inactivation of the mutant XLA/BTK allele in B-lymphocytes but not in
T-lymphocytes), highlights the role of functional cellular selection (Fearon et al .,
1987; reviewed in Belmont, 1996). In addition, skewing appears more common in
older women, which suggests the contribution of environmental factors throughout
their lifespan (Busque et al ., 1996; Gale et al ., 1997; Sharp et al ., 2000). In
this regard, X-inactivation seems to remain quite stable over many years during
earlier ages (Sandovici et al ., 2004) (Figure 1b). Many older females, however,
exhibit substantial changes over the timescales at which younger females do
not exhibit changes (Sandovici et al ., 2004). In this regard, we have speculated
(Sandovici et al ., 2004) that acquired skewing of X-inactivation in older females
may result from discontinuous or catastrophic processes that result in decreased
numbers of stem cells or an age-related tendency toward bone marrow clonality or
myelodysplasia.
Additionally, preference for the inactivation of a particular X chromosome can
have a completely different origin compared to the selection for particular clonal
cell populations or against disadvantageous mutations. Several studies in human and
mice have shown that preference for X-inactivation can be heritable and genetically
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6 Epigenetics
controlled (Cattanach and Isaacson, 1967; Naumova et al ., 1996, 1998; Plenge
et al ., 1997) In the mouse, the X-controlling element (Xce) is well known for its
participation in the X-inactivation choice, so chromosomes carrying different alleles
of Xce have different probabilities of being inactivated (Cattanach and Isaacson,
1967) (Figure 1c). Additional autosomal loci also participate in the genetic control
of the choice of the X chromosome to be inactivated in mice (Chadwick and
Willard, 2005; Percec et al ., 2002, 2003). Moreover, parent-of-origin effects have
also been observed in both mice (Takagi and Sasaki, 1975) and humans (Chadwick
and Willard, 2005).
Stochastic, environmental, and genetic factors result in variability in X-chromosome inactivation and, consequently, generate a gamut of phenotypes for each
of the X-linked genes, with multiple implications. The relative abundance of
transcripts of each allele of any gene subject to X-inactivation reflects the fraction of
cells with each of the two chromosomes active, as well as any allelic differences in
expression that are intrinsic to specific alleles. Variations in such relative expression
result in the spectrum of phenotypes observed in the population. For instance,
a correlation between X-inactivation patterns and meiotic recombination levels
(genomewide) has been described in female mice (de la Casa-Esperon et al .,
2002). The biological importance of this trait (recombination levels) in the human
population cannot be overestimated as it is a major determinant of female fecundity
and reproductive lifespan. If recombination levels are controlled by gene/s in the
X chromosome, then levels of recombination can change accordingly with the
relative expression of different alleles of such gene/s. Because this is only one of
the numerous genes in the X chromosome, the phenotypic diversity generated by
similar phenomena related to X-inactivation processes is expected to be large in
female mammals.
Similarly, epigenetic variability between individuals at multiple autosomal loci
can be the result of multiple processes. Since erasure and establishment of
epigenetic marks is a dynamic process that occurs during the lifespan of organisms,
especially during gametogenesis and embryogenesis (reviewed in Latham, 1999;
Mann and Bartolomei, 2002; Article 33, Epigenetic reprogramming in germ cells
and preimplantation embryos, Volume 1), there is ample room for stochastic
factors to contribute to the diversity of patterns observed. Environmental effects
have also been described. Nutritional factors can induce epigenetic modifications
such as changes in the expression of imprinted genes; moreover, maternal diet can
affect the methylation status of transposable elements and the expression of nearby
genes in mice (reviewed in Waterland and Jirtle, 2004). Examples of environmental
effects have also been reported in rats, in which variations in maternal care behavior
result in epigenetic changes in the offspring at the level of histone acetylation and
DNA methylation of the consensus sequence for the NGFI-A transcription factor
of the glucocorticoid receptor gene. Consequently, expression of this gene in the
hippocampus can be modified by maternal care, which might be the basis for the
changes in stress response observed in this gene in the offspring (reviewed in
Fish et al ., 2004). Environmental effects could be also the basis for the changes
observed in epigenetic marks over time. DNA methylation patterns change with
aging in a complex fashion, although overall hypomethylation has been observed
in most vertebrate tissues (Mays-Hoopes et al ., 1986; Richardson, 2003). For
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instance, changes in the methylation profile of the c-myc proto-oncogene have
been described during the aging process of mice. Because this is a gene involved
in many tumor processes, similar temporal alterations of epigenetic marks might
be part of the basis of the increasing incidence of cancer with age (Ono et al .,
1986, 1989).
Finally, epigenetic diversity can be the result of heritable variants that affect
the formation or stability of epigenetic marks. It has been observed that allelic
differences in the expression of several genes are transmitted in families, although
the patterns of transmission are variable (Pastinen et al ., 2004; Yan et al ., 2002). In
some instances, the transmission of allelic imbalance is compatible with Mendelian
inheritance, and even associated with transmission of particular polymorphisms
(haplotypes), suggesting the participation of cis-acting elements in the regulation
of allelic expression (Yan et al ., 2002), whether they are of genetic or epigenetic
origin. In fact, studies showing transmission of de novo induced methylation
changes indicate that chromatin modifications, per se, are heritable (Kakutani
et al ., 1999; Stokes et al ., 2002). Moreover, abnormal methylation patterns at the
differentially methylated regions of the IGF2/H19 and IGF2R imprinted genes have
been found to cluster in families (Sandovici et al ., 2003). Also, methylation levels
at particular Alu repeated sequences show interindividual differences when the
insertions were paternally versus maternally transmitted (Sandovici et al ., 2005).
In the case of imprinting defects, epimutations in an imprinting control region of
human chromosome 15 have been associated with a substantial percentage of cases
of the neurodevelopmental disorders Angelman and Prader–Willi syndromes (see
Article 29, Imprinting in Prader–Willi and Angelman syndromes, Volume 1).
Recent studies have shown that both cis- and trans-acting factors seem to increase
the risk of conceiving a child with Angelman syndrome (AS) (Zogel et al ., 2006).
Trans-acting genetic elements have also been involved in changes in the imprinting
status of the Dlk1 gene in mouse brain (Croteau et al ., 2005). In this case,
reactivation of the normally silent maternal allele correlates with the methylation
status of a differentially methylated region. Therefore, epigenetic information
constitutes a code superimposed on the genetic information, thereby increasing
phenotypic diversity. Much future research will no doubt focus on determining
whether epigenetic variation makes a significant contribution to common “complex
genetic disorders”, such as diabetes, hypertension, schizophrenia, Alzheimer’s
disease and the like, in humans.
4. Epigenetic variation: what are the consequences?
Phenotypic diversity is the direct consequence of much epigenetic variation. As
we mentioned before, epigenetic modifications can result in allelic expression
imbalance within (differential expression levels) or between cells (monoallelic and
mosaic expression). This, in turn, can result in phenotypic differences between cells,
tissues, and/or individuals. The most obvious example is that of monozygotic twins:
although genetically identical, numerous phenotypic differences appear during their
life span. The same is true at the epigenetic level: recent studies have shown that
differences in DNA methylation and histone acetylation between twins are present
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throughout the genome (Fraga et al ., 2005). Therefore, epigenetic differences could
be the basis of many phenotypic discordances observed between twins, including
their susceptibility to complex diseases (Wong et al ., 2005).
4.1. Epigenetic variation and disease
Epigenetic variation is particularly important for genes involved in diseases. For
instance, the fragile-X syndrome of mental retardation is associated with an
expansion in the number of CGG repeats in the promoter and 5 untranslated region
of the FMR1 gene on chromosome X. This expansion results in hypermethylation of
the region and silencing of the FMR1 gene (Hansen et al ., 1992). Short expansions
(premutations) do not have apparent phenotypic effects, while long expansions are
observed in affected individuals. Notably, the severity of the disease ranges from
severe mental retardation to only mild learning disabilities. It is possible that the
observed gamut of symptoms depends, at least in part, on epigenetic differences,
because variability in methylation in this region has been observed between and
within individuals (Genc et al ., 2000; Stoger et al ., 1997) and changes in the
CGG repeat length might also result in additional chromatin and transcriptional
modifications.
Another interesting example of mosaicism has been observed in a small group
of AS patients, in whom an imprinting defect silences the maternal copy of the
UBE3A gene. However, some of these patients show mosaic maternal expression
and methylation of this gene, which, again, suggests the possibility of an epigenetic
effect on the observed variability in the severity of clinical symptoms (Nazlican
et al ., 2004).
Cancer has also been associated with epigenetic alterations, such as losses and
gains of methylation and LOI (Feinberg et al ., 1988, 2002; Cui et al ., 2003; Jones
and Baylin, 2002; Nakagawa et al ., 2001). Interestingly, some of these alterations
are also observed in normal tissues of the same individuals, as highlighted by
the gain of DNA methylation in the imprinting control region upstream of H19
in human Wilms tumors and in the non-neoplastic kidney parenchyma adjacent
to these tumors (Cui et al ., 1998, 2003; Moulton et al ., 1994). Hence, epigenetic
variation between individuals is probably involved in susceptibility to develop
cancer as well as other genetic diseases. Moreover, since heritable epigenetic
variation has been observed in many instances, it can actually play an important role
in quantitative trait variation, and selection acting on such epialleles might result
in rapid phenotypic changes, making it a formidable force in evolution (Rutherford
and Henikoff, 2003; Sollars et al ., 2003).
4.2. Epigenetic variation and development
Epigenetic variation also has important consequences in development and
differentiation. A potentially important example of epigenetic changes as a result
of environmental effects is the effects of culture conditions on the expression of
imprinted genes in mouse embryos. It has been shown that some culture media
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perturbs gene expression and results in aberrant methylation and expression of
imprinted genes (Doherty et al ., 2000; Mann et al ., 2004; Rinaudo and Schultz,
2004). Although some of these abnormalities can be restored in the embryo proper
(Mann et al ., 2004), many persist in the extraembryonic tissues and can potentially
affect the development of the embryo. In fact, several epidemiological studies
suggest that assisted reproductive technologies (ART) might result in an increased
frequency of diseases caused by imprinting defects, such as AS and BeckwithWiedemann syndrome (BWS) (Article 30, Beckwith–Wiedemann syndrome,
Volume 1).
Despite the many reassuring reports on the safety of ART, there have been a
small number of recent reports suggesting that ART children may be at increased
risk for rare congenital malformation syndromes that are related to defects in
genome imprinting (Cox et al ., 2002; DeBaun et al ., 2003; Halliday et al ., 2004;
Horsthemke et al ., 2003; Niemitz et al ., 2004; Olivennes et al ., 2001; Orstavik
et al ., 2003). At least three children conceived by intracytoplasmic sperm injection
(ICSI) have been diagnosed with AS (Horsthemke et al ., 2003; Orstavik et al .,
2003) and at least 28 ART children (both in vitro fertilization (IVF) and ICSI
cases) have been diagnosed with BWS (Boerrigter et al ., 2002; Bonduelle et al .,
2002; DeBaun et al ., 2003; Gicquel et al ., 2003; Halliday et al ., 2004; Koudstaal
et al ., 2000; Maher et al ., 2003; Olivennes et al ., 2001; Sutcliffe et al ., 1995).
Because both AS and BWS are rare disorders (each affects approximately 1 in
15 000 children (Nicholls et al ., 1998)), the appearance of even small numbers of
cases is unexpected except among a large sample of births. Therefore, the current
data strongly suggests that there is an association between increased risk for AS and
BWS and ART. With respect to BWS, the number of affected individuals observed
is estimated to be up to nine times the expected incidence (Halliday et al ., 2004).
The epidemiological assessment that ART may lead to an increase in the
frequency of defective genome imprints is also supported by biochemical
characterization of alleles at the relevant disease loci. All three cases of AS show
allelic DNA methylation patterns characteristic of a sporadic imprinting defect at
the AS locus (i.e., complete or mosaic absence of methylation on both maternal
and paternal alleles (Horsthemke et al ., 2003; Orstavik et al ., 2003)). None of the
patients has a cytogenetically visible alteration of chromosome 15 (which occurs in
70% of all AS cases (Nicholls et al ., 1998)) and none has a detectable microdeletion
at the imprinting center, suggesting that all three cases are due to sporadic, primary,
epigenetic defects rather than genetic changes. Given that such imprinting defects
account for less than 5% of all AS cases (Buiting et al ., 2001, 2003; Nicholls
et al ., 1998), there is at least a suspicion that all three cases occurring in patients
following ICSI are of this type.
The case for the presence of primary epigenetic defects in the majority of the
BWS patients found among ART children is also supported by molecular analyses
of alleles at the BWS locus on chromosome 11. Nineteen of the 24 patients
have been analyzed for “loss of imprinting” (“LOI”; defined, in this context, as
transcription of both maternal and paternal alleles; or the specific changes in DNA
methylation that track with this phenomenon and provide a more robust marker in
clinical samples) at one or more imprinted genes within the BWS locus and 13
of the 19 cases showed LOI at either KCNQ10 T1 (DeBaun et al ., 2003; Gicquel
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et al ., 2003; Maher et al ., 2003) or H19/IGF2 (DeBaun et al ., 2003). With the
addition of the BWS patients described by Halliday et al ., 2004, 16 out of a total
of 22 cases examined showed LOI. Although imprinting defects are more common
in BWS than in AS, LOI still appears to be overrepresented among BWS cases in
ART children and ART is, in turn, overrepresented among BWS cases.
4.3. Epigenetic variation diversity
During the last several years, there has been a dramatic increase in the number
of studies attempting to elucidate the patterns and interrelationship between
DNA methylation, histone modifications, noncoding RNAs, binding of nonhistone
chromatin proteins, nuclear positioning and interactions, and so on, which are part
of the “epigenetic code” (Article 27, The histone code and epigenetic inheritance,
Volume 1). Alterations of the chromatin configuration can affect interactions
between DNA regions, between chromosomes, and with other molecules. Most of
the studies in epigenetic variation have been focused on the different mechanisms
and effects on gene expression and its phenotypic consequences, including allelic
differences and disease, enhancers and insulators, trans-sensing and paramutation,
long-range interactions and nuclear colocation, and so on. However, epigenetic
changes have also been found to affect many other chromosomal functions (see
the following text). A classical example is the centromere, in which multiple
chromatin modifications and proteins play a major role in binding to the poles
of the spindle and promoting chromosome segregation. Interestingly, epigenetic
changes can generate new domains with similar properties (neocentromeres)
that affect the segregation of chromosomes during mitosis and meiosis (PardoManuel de Villena and Sapienza, 2001; Rhoades and Dempsey, 1966; Warburton,
2004). Consequently, changes in the segregation of chromosomes or chromatids
can favor the transmission of particular alleles to the next generations, with
important consequences in evolution and disease (Pardo-Manuel de Villena and
Sapienza, 2001).
Another example of a biochemical process for which there is a strong epigenetic
effect is asynchronous DNA replication. Asynchronous replication is characteristic
of regions containing monoallelically expressed genes (Mostoslavsky et al ., 2001;
Simon et al ., 1999) and, therefore, epigenetic differences seem to be the basis for
the differential replication between homologs at such regions. Consequently, these
chromosomal regions are interesting examples of how epigenetic modifications
of chromosomal regions have not one but multiple effects (on replication and
expression). In addition, a recent survey of asynchronously replicated regions have
found that they are located in close proximity to areas of tandem gene duplication
(Gimelbrant and Chess, 2006) – although whether such epigenetic marks play a
role in chromosome stability in regions of duplications remains to be determined.
Meiotic pairing and recombination constitute another example of a cellular
process in which epigenetic marking appears to play an important role. Functional
and epigenetic differences between paternal and maternal chromosomes are a
common observation in sexually reproducing organisms (reviewed in de la CasaEsperon and Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000). However, only
Specialist Review
a few of such differences have been associated with imprinted gene expression.
Consequently, it has been postulated that parent-of-origin epigenetic differences
share a common origin and function in all sexually reproducing organisms: to
allow the recognition (and distinction) between homologous chromosomes during
the processes of recombination and repair (de la Casa-Esperon and Sapienza,
2003; Pardo-Manuel de Villena et al ., 2000). Indeed, a recent study has shown
that DNA methylation has a role in early meiotic stages: mice deficient in the
DNA methyltransferase 3-like (Dnmt3L) gene are sterile and display abnormal
chromosome synapsis during meiosis (Bourc’his and Bestor, 2004). Curiously,
normal expression of Dnmt3L occurs not in the meiotic cells, but in their precursors.
Hence, the epigenetic signals must be inherited through multiple cell divisions. Such
epigenetic signals are observed as DNA methylation of retrotransposons, which
appear demethylated in Dnmt3L knockout male germ cells. While methylation
participates in the normal silencing of mobile elements, retrotransposons are
transcribed in the mutant mice. Therefore, Dnmt3L mutant mice represent an
example of how epigenetic changes can not only affect transcription but can
also reshape the genome by affecting synapsis and allowing the mobilization of
retrotransposons into new locations, with multiple consequences. Consequently,
studies of epigenetic variation cannot be restricted to effects on gene expression,
because it can also modulate many other chromosome functions (de la CasaEsperon and Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000; Sandovici
et al ., 2005).
5. Conclusions
When discussing epigenetic variation, it is important to remember that we know
little about either the underlying mechanisms or the consequences. To mention a
few recent examples, studies on the viable yellow allele of the mouse agouti locus
(Avy ) have shown that the expression of the agouti gene is correlated with the
methylation status of upstream sequences (Article 36, Variable expressivity and
epigenetics, Volume 1). Interestingly, epigenetic inheritance at this locus is not due
to such methylation marks, because they are erased during embryonic development
(Blewitt et al ., 2006). Therefore, other epigenetic marks are responsible for the
transmission of this epiallele to the offspring. In this review, although we have
mostly mentioned examples of variability in methylation (because it has been the
most frequently studied epigenetic mark in mammals and is the first subject of the
Human Epigenome Project (Eckhardt et al ., 2004)), we hope that current and future
studies will bring to light epigenetic variation at many other levels. For instance,
studies of the effects of histone tail modifications at multiple amino acid residues
are an expanding field, because the spectrum of modifications and residues affected
continues to grow (Article 27, The histone code and epigenetic inheritance,
Volume 1). In addition, new epigenetic marks and inheritance modes are likely
to be discovered. For instance, the role of small RNAs on epigenetic changes
has become prominent since the discovery of RNA interference in Caenorhabditis
elegans (Fire et al ., 1998). Recent studies have revealed striking new roles for
RNA in non-Mendelian epigenetic inheritance, similar to paramutation in plants.
11
12 Epigenetics
The homozygous wild-type progeny of mice that are heterozygous for a mutation in
the Kit gene (Rassoulzadegan et al ., 2006) are found to exhibit the white spotting
phenotype that is characteristic of mice that carry a Kit mutation. Elaboration of
this phenotype is related to the zygotic inheritance of abnormally processed RNAs
of the normal allele.
A realistic description of the scale of epigenetic variation is hampered by
the diversity of causes and consequences and because the mechanism by which
many epigenetic marks are heritable remains obscure. An increasing number of
studies are aiming to integrate profiles from different epigenetic marks and gene
expression patterns of particular chromosomal regions, in order to better understand
the possibilities of variations on the epigenetic code. The complexity and diversity
of the epigenetic marks and their implications poses a tremendous challenge, but
understanding the nature of the immense phenotypic diversity that surrounds us
makes it worth the effort.
Further Readings
Kochanek S, Renz D and Doerfler W (1994) Variability in allelic DNA methylation in
spermatozoa. Human Genetics, 94, 203–206.
Maher ER (2005) Imprinting and assisted reproductive technology. Human Molecular Genetics,
14(Spec No. 1), R133–R138.
References
Belmont JW (1996) Genetic control of X inactivation and processes leading to X-inactivation
skewing. American Journal of Human Genetics, 58, 1101–1108.
Blewitt ME, Vickaryous NK, Paldi A, Koseki H and Whitelaw E (2006) Dynamic reprogramming
of DNA methylation at an epigenetically sensitive allele in mice. PLoS Genetics, 2, e49.
Boerrigter PJ, de Bie JJ, Mannaerts BM, van Leeuwen BP and Passier-Timmermans DP (2002)
Obstetrical and neonatal outcome after controlled ovarian stimulation for IVF using the GnRH
antagonist ganirelix. Human Reproduction, 17, 2027–2034.
Bonduelle M, Liebaers I, Deketelaere V, Derde MP, Camus M, Devroey P and Van Steirteghem A
(2002) Neonatal data on a cohort of 2889 infants born after ICSI (1991–1999) and of 2995
infants born after IVF (1983–1999). Human Reproduction, 17, 671–694.
Bourc’his D and Bestor TH (2004) Meiotic catastrophe and retrotransposon reactivation in male
germ cells lacking Dnmt3L. Nature, 431, 96–99.
Buiting K, Barnicoat A, Lich C, Pembrey M, Malcolm S and Horsthemke B (2001) Disruption
of the bipartite imprinting center in a family with Angelman syndrome. American Journal of
Human Genetics, 68, 1290–1294.
Buiting K, Gross S, Lich C, Gillessen-Kaesbach G, el-Maarri O and Horsthemke B (2003)
Epimutations in Prader-Willi and Angelman syndromes: a molecular study of 136 patients
with an imprinting defect. American Journal of Human Genetics, 72, 571–577.
Bunzel R, Blumcke I, Cichon S, Normann S, Schramm J, Propping P and Nothen MM (1998)
Polymorphic imprinting of the serotonin-2A (5-HT2A) receptor gene in human adult brain.
Molecular Brain Research, 59, 90–92.
Busque L, Mio R, Mattioli J, Brais E, Blais N, Lalonde Y, Maragh M and Gilliland DG (1996)
Nonrandom X-inactivation patterns in normal females: lyonization ratios vary with age. Blood ,
88, 59–56.
Carrel L and Willard HF (2005) X-inactivation profile reveals extensive variability in X-linked
gene expression in females. Nature, 434, 400–404.
Specialist Review
de la Casa-Esperon E, Loredo-Osti JC, Pardo-Manuel de Villena F, Briscoe TL, Malette JM,
Vaughan JE, Morgan K and Sapienza C (2002) X chromosome effect on maternal
recombination and meiotic drive in the mouse. Genetics, 161, 1651–1659.
de la Casa-Esperon E and Sapienza C (2003) Natural selection and the evolution of genome
imprinting. Annual Review of Genetics, 37, 349–370.
Cattanach BM and Isaacson JH (1967) Controlling elements in the mouse X chromosome.
Genetics, 57, 331–346.
Chadwick LH and Willard HF (2005) Genetic and parent-of-origin influences on X chromosome
choice in Xce heterozygous mice. Mammalian Genome, 16, 691–699.
Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL and Horsthemke B (2002) Intracytoplasmic
sperm injection may increase the risk of imprinting defects. American Journal of Human
Genetics, 71, 162–164.
Croteau S, Roquis D, Charron MC, Frappier D, Yavin D, Loredo-Osti JC, Hudson TJ and
Naumova AK (2005) Increased plasticity of genomic imprinting of Dlk1 in brain is due
to genetic and epigenetic factors. Mammalian Genome, 16, 127–135.
Cui H, Cruz-Correa M, Giardiello FM, Hutcheon DF, Kafonek DR, Brandenburg S, Wu Y, He X,
Powe NR and Feinberg AP (2003) Loss of IGF2 imprinting: a potential marker of colorectal
cancer risk. Science, 299, 1753–1755.
Cui H, Horon IL, Ohlsson R, Hamilton SR and Feinberg AP (1998) Loss of imprinting in
normal tissue of colorectal cancer patients with microsatellite instability. Nature Medicine,
4, 1276–1280.
Dao D, Frank D, Qian N, O’Keefe D, Vosatka RJ, Walsh CP and Tycko B (1998) IMPT1, an
imprinted gene similar to polyspecific transporter and multi-drug resistance genes. Human
Molecular Genetics, 7, 597–608.
Das OP and Messing J (1994) Variegated phenotype and developmental methylation changes of
a maize allele originating from epimutation. Genetics, 136, 1121–1141.
DeBaun MR, Niemitz EL and Feinberg AP (2003) Association of in vitro fertilization with
Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. American
Journal of Human Genetics, 72, 156–160.
Disteche CM (1995) Escape from X inactivation in human and mouse. Trends in Genetics, 11,
17–22.
Doherty AS, Mann MR, Tremblay KD, Bartolomei MS and Schultz RM (2000) Differential
effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biology
of Reproduction, 62, 1526–1535.
Eckhardt F, Beck S, Gut IG and Berlin K (2004) Future potential of the Human Epigenome
Project. Expert Review of Molecular Diagnostics, 4, 609–618.
Fearon ER, Winkelstein JA, Civin CI, Pardoll DM and Vogelstein B (1987) Carrier detection
in X-linked agammaglobulinemia by analysis of X-chromosome inactivation. New England
Journal of Medicine, 316, 427–431.
Feinberg AP, Cui H and Ohlsson R (2002) DNA methylation and genomic imprinting: insights
from cancer into epigenetic mechanisms. Seminars in Cancer Biology, 12, 389–398.
Feinberg AP, Gehrke CW, Kuo KC and Ehrlich M (1988) Reduced genomic 5-methylcytosine
content in human colonic neoplasia. Cancer Research, 48, 1159–1161.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE and Mello CC (1998) Potent and
specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391,
806–811.
Fish EW, Shahrokh D, Bagot R, Caldji C, Bredy T, Szyf M and Meaney MJ (2004) Epigenetic
programming of stress responses through variations in maternal care. Annual New York
Academy of Sciences, 1036, 167–180.
Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, Ballestar ML, Heine-Suner D, Cigudosa JC,
Urioste M, Benitez J, et al (2005) Epigenetic differences arise during the lifetime of
monozygotic twins. Proceedings of the National Academy of Sciences of the United States
of America, 102, 10604–10609.
Gale RE, Fielding AK, Harrison CN and Linch DC (1997) Acquired skewing of X-chromosome
inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age.
British Journal of Haematology, 98, 512–519.
13
14 Epigenetics
Genc B, Muller-Hartmann H, Zeschnigk M, Deissler H, Schmitz B, Majewski F, von Gontard A
and Doerfler W (2000) Methylation mosaicism of 5 -(CGG)(n)-3 repeats in fragile X,
premutation and normal individuals. Nucleic Acids Research, 28, 2141–2152.
Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A and Le Bouc Y (2003) In vitro
fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal
imprinting of the KCN1OT gene. American Journal of Human Genetics, 72, 1338–1341.
Gimelbrant AA and Chess A (2006) An epigenetic state associated with areas of gene duplication.
Genome Research, 16, 723–729.
Halliday J, Oke K, Breheny S, Algar E and Amor DJ (2004) Beckwith-Wiedemann syndrome
and IVF: a case-control study. American Journal of Human Genetics, 75, 526–528.
Hansen RS, Gartler SM, Scott CR, Chen SH and Laird CD (1992) Methylation analysis of CGG
sites in the CpG island of the human FMR1 gene. Human Molecular Genetics, 1, 571–578.
Horsthemke B, Nazlican H, Husing J, Klein-Hitpass L, Claussen U, Michel S, Lich C, GillessenKaesbach G and Buiting K (2003) Somatic mosaicism for maternal uniparental disomy 15 in
a girl with Prader-Willi syndrome: confirmation by cell cloning and identification of candidate
downstream genes. Human Molecular Genetics, 12, 2723–2732.
Jones PA and Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nature
Reviews Genetics, 3, 415–428.
Kakutani T, Munakata K, Richards EJ and Hirochika H (1999) Meiotically and mitotically stable
inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana.
Genetics, 151, 831–838.
Koudstaal J, Braat DD, Bruinse HW, Naaktgeboren N, Vermeiden JP and Visser GH (2000)
Obstetric outcome of singleton pregnancies after IVF: a matched control study in four Dutch
university hospitals. Human Reproduction, 15, 1819–1825.
Latham KE (1999) Epigenetic modification and imprinting of the mammalian genome during
development. Current Topics in Developmental Biology, 43, 1–49.
Lin W, Yang HH and Lee MP (2005) Allelic variation in gene expression identified through
computational analysis of the dbEST database. Genomics, 86, 518–527.
Lo HS, Wang Z, Hu Y, Yang HH, Gere S, Buetow KH and Lee MP (2003) Allelic variation in
gene expression is common in the human genome. Genome Research, 13, 1855–1862.
Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, Macdonald F, Sampson JR,
Barratt CL, Reik W, et al (2003) Beckwith-Wiedemann syndrome and assisted reproduction
technology (ART). Journal of Medical Genetics, 40, 62–64.
Mann MR and Bartolomei MS (2002) Epigenetic reprogramming in the mammalian embryo:
struggle of the clones. Genome Biology, 3, Reviews 1003.
Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM and Bartolomei MS (2004)
Selective loss of imprinting in the placenta following preimplantation development in culture.
Development, 131, 3727–3735.
Mays-Hoopes L, Chao W, Butcher HC and Huang RC (1986) Decreased methylation of the major
mouse long interspersed repeated DNA during aging and in myeloma cells. Developmental
Genetics, 7, 65–73.
McMinn J, Wei M, Sadovsky Y, Thaker HM and Tycko B (2006) Imprinting of PEG1/MEST
isoform 2 in human placenta. Placenta, 27, 119–126.
Migeon BR, Axelman J and Beggs AH (1988) Effect of ageing on reactivation of the human
X-linked HPRT locus. Nature, 335, 93–96.
Mostoslavsky R, Singh N, Tenzen T, Goldmit M, Gabay C, Elizur S, Qi P, Reubinoff BE,
Chess A, Cedar H, et al (2001) Asynchronous replication and allelic exclusion in the immune
system. Nature, 414, 221–225.
Moulton T, Crenshaw T, Hao Y, Moosikasuwan J, Lin N, Dembitzer F, Hensle T, Weiss L,
McMorrow L, Loew T, et al (1994) Epigenetic lesions at the H19 locus in Wilms’ tumour
patients. Nature Genetics, 7, 440–447.
Nakagawa H, Chadwick RB, Peltomaki P, Plass C, Nakamura Y and de La Chapelle A (2001)
Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in
a core region of H19-associated CTCF-binding sites in colorectal cancer. Proceedings of the
National Academy of Sciences of the United States of America, 98, 591–596.
Specialist Review
Naumova AK, Olien L, Bird LM, Smith M, Verner AE, Leppert M, Morgan K and Sapienza C
(1998) Genetic mapping of X-linked loci involved in skewing of X chromosome inactivation
in the human. European Journal of Human Genetics, 6, 552–562.
Naumova AK, Plenge RM, Bird LM, Leppert M, Morgan K, Willard HF and Sapienza C (1996)
Heritability of X chromosome-inactivation phenotype in a large family. American Journal of
Human Genetics, 58, 1111–1119.
Nazlican H, Zeschnigk M, Claussen U, Michel S, Boehringer S, Gillessen-Kaesbach G, Buiting K
and Horsthemke B (2004) Somatic mosaicism in patients with Angelman syndrome and an
imprinting defect. Human Molecular Genetics, 13, 2547–2555.
Nicholls RD, Saitoh S and Horsthemke B (1998) Imprinting in Prader-Willi and Angelman
syndromes. Trends in Genetics, 14, 194–200.
Niemitz EL, DeBaun MR, Fallon J, Murakami K, Kugoh H, Oshimura M and Feinberg AP
(2004) Microdeletion of LIT1 in familial Beckwith-Wiedemann syndrome. American Journal
of Human Genetics, 75, 844–849.
Olivennes F, Mannaerts B, Struijs M, Bonduelle M and Devroey P (2001) Perinatal outcome
of pregnancy after GnRH antagonist (ganirelix) treatment during ovarian stimulation for
conventional IVF or ICSI: a preliminary report. Human Reproduction, 16, 1588–1591.
Ono T, Takahashi N and Okada S (1989) Age -associated changes in DNA methylation and
mRNA level of the c-myc gene in spleen and liver of mice. Mutation Research, 219, 39–50.
Ono T, Tawa R, Shinya K, Hirose S and Okada S (1986) Methylation of the c-myc gene changes
during aging process of mice. Biochemical and Biophysical Research Communication, 139,
1299–1304.
Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O and Buiting K (2003)
Another case of imprinting defect in a girl with Angelman syndrome who was conceived by
intracytoplasmic semen injection. American Journal of Human Genetics, 72, 218–219.
Pagani F, Toniolo D and Vergani C (1990) Stability of DNA methylation of X-chromosome genes
during aging. Somatic Cell and Molecular Genetics, 16, 79–84.
Paldi A (2003) Stochastic gene expression during cell differentiation: order from disorder? Cell
and Molecular Life Sciences, 60, 1775–1778.
Pant PV, Tao H, Beilharz EJ, Ballinger DG, Cox DR and Frazer KA (2006) Analysis of allelic
differential expression in human white blood cells. Genome Research, 16, 331–339.
Pardo-Manuel de Villena F, de la Casa-Esperon E and Sapienza C (2000) Natural selection and
the function of genome imprinting: beyond the silenced minority. Trends in Genetics, 16,
573–579.
Pardo-Manuel de Villena F and Sapienza C (2001) Nonrandom segregation during meiosis: the
unfairness of females. Mammalian Genome, 12, 331–339.
Pastinen T, Sladek R, Gurd S, Sammak A, Ge B, Lepage P, Lavergne K, Villeneuve A, Gaudin T,
Brandstrom H, et al (2004) A survey of genetic and epigenetic variation affecting human gene
expression. Physiological Genomics, 16, 184–193.
Percec I, Plenge RM, Nadeau JH, Bartolomei MS and Willard HF (2002) Autosomal dominant
mutations affecting X inactivation choice in the mouse. Science, 296, 1136–1139.
Percec I, Thorvaldsen JL, Plenge RM, Krapp CJ, Nadeau JH, Willard HF and Bartolomei MS
(2003) An N-ethyl-N-nitrosourea mutagenesis screen for epigenetic mutations in the mouse.
Genetics, 164, 1481–1494.
Plenge RM, Hendrich BD, Schwartz C, Arena JF, Naumova A, Sapienza C, Winter RM and
Willard HF (1997) A promoter mutation in the XIST gene in two unrelated families with
skewed X-chromosome inactivation. Nature Genetics, 17, 353–356.
Rangwala SH, Elumalai R, Vanier C, Ozkan H, Galbraith DW and Richards EJ (2006) Meiotically
stable natural epialleles of sadhu, a novel arabidopsis retroposon. PLoS Genetics, 2, e36.
Rassoulzadegan M, Grandjean V, Gounon P, Vincent S, Gillot I and Cuzin F (2006) RNAmediated non-mendelian inheritance of an epigenetic change in the mouse. Nature, 441,
469–474.
Rhoades MM and Dempsey E (1966) The effect of abnormal chromosome 10 on preferential
segregation and crossing over in maize. Genetics, 53, 989–1020.
Richardson B (2003) Impact of aging on DNA methylation. Ageing Research Reviews, 2,
245–261.
15
16 Epigenetics
Riddle NC and Richards EJ (2002) The control of natural variation in cytosine methylation in
Arabidopsis. Genetics, 162, 355–363.
Riddle NC and Richards EJ (2005) Genetic variation in epigenetic inheritance of ribosomal RNA
gene methylation in Arabidopsis. Plant Journal , 41, 524–532.
Rinaudo P and Schultz RM (2004) Effects of embryo culture on global pattern of gene expression
in preimplantation mouse embryos. Reproduction, 128, 301–311.
Rutherford SL and Henikoff S (2003) Quantitative epigenetics. Nature Genetics, 33, 6–8.
Sakatani T, Wei M, Katoh M, Okita C, Wada D, Mitsuya K, Meguro M, Ikeguchi M, Ito H,
Tycko B, et al (2001) Epigenetic heterogeneity at imprinted loci in normal populations.
Biochemical and Biophysical Research Communication, 283, 1124–1130.
Sandovici I, Kassovska-Bratinova S, Loredo-Osti JC, Leppert M, Suarez A, Stewart R, Bautista
FD, Schiraldi M and Sapienza C (2005) Interindividual variability and parent of origin DNA
methylation differences at specific human Alu elements. Human Molecular Genetics, 14,
2135–2143.
Sandovici I, Leppert M, Hawk PR, Suarez A, Linares Y and Sapienza C (2003) Familial
aggregation of abnormal methylation of parental alleles at the IGF2/H19 and IGF2 R
differentially methylated regions. Human Molecular Genetics, 12, 1569–1578.
Sandovici I, Naumova AK, Leppert M, Linares Y and Sapienza C (2004) A longitudinal study
of X-inactivation ratio in human females. Human Genetics, 115, 387–392.
Sharp A, Robinson D and Jacobs P (2000) Age- and tissue-specific variation of X chromosome
inactivation ratios in normal women. Human Genetics, 107, 343–349.
Simon I, Tenzen T, Reubinoff BE, Hillman D, McCarrey JR and Cedar H (1999) Asynchronous
replication of imprinted genes is established in the gametes and maintained during
development. Nature, 401, 929–932.
Sollars V, Lu X, Xiao L, Wang X, Garfinkel MD and Ruden DM (2003) Evidence for an epigenetic
mechanism by which Hsp90 acts as a capacitor for morphological evolution. Nature Genetics,
33, 70–74.
Stoger R, Kajimura TM, Brown WT and Laird CD (1997) Epigenetic variation illustrated by DNA
methylation patterns of the fragile-X gene FMR1. Human Molecular Genetics, 6, 1791–1801.
Stokes TL, Kunkel BN and Richards EJ (2002) Epigenetic variation in Arabidopsis disease
resistance. Genes and Development, 16, 171–182.
Sutcliffe AG, D’Souza SW, Cadman J, Richards B, McKinlay IA and Lieberman B (1995) Minor
congenital anomalies, major congenital malformations and development in children conceived
from cryopreserved embryos. Human Reproduction, 10, 3332–3337.
Takagi N and Sasaki M (1975) Preferential inactivation of the paternally derived X chromosome
in the extraembryonic membranes of the mouse. Nature, 256, 640–642.
Warburton PE (2004) Chromosomal dynamics of human neocentromere formation. Chromosome
Research, 12, 617–626.
Wareham KA, Lyon MF, Glenister PH and Williams ED (1987) Age related reactivation of an
X-linked gene. Nature, 327, 725–727.
Waterland RA and Jirtle RL (2004) Early nutrition, epigenetic changes at transposons and
imprinted genes, and enhanced susceptibility to adult chronic diseases. Nutrition, 20, 63–68.
Williams BR and Wu CT (2004) Does random X-inactivation in mammals reflect a random choice
between two X chromosomes? Genetics, 167, 1525–1528.
Wolff GL, Kodell RL, Moore SR and Cooney CA (1998) Maternal epigenetics and methyl
supplements affect agouti gene expression in Avy/a mice. FASEB Journal , 12, 949–957.
Wong AH, Gottesman II and Petronis A (2005) Phenotypic differences in genetically identical
organisms: the epigenetic perspective. Human Molecular Genetics, 14(Spec No. 1), R11–R18.
Xu Y, Goodyer CG, Deal C and Polychronakos C (1993) Functional polymorphism in the parental
imprinting of the human IGF2 R gene. Biochemical and Biophysical Research Communication,
197, 747–754.
Yan H, Yuan W, Velculescu VE, Vogelstein B and Kinzler KW (2002) Allelic variation in human
gene expression. Science, 297, 1143.
Zogel C, Bohringer S, Gross S, Varon R, Buiting K and Horsthemke B (2006) Identification of
cis- and trans-acting factors possibly modifying the risk of epimutations on chromosome 15.
European Journal of Human Genetics, 14, 752–758.