Review

A twin approach to unraveling

epigenetics
Jordana T. Bell1,2 and Tim D. Spector1
1

Department of Twin Research and Genetic Epidemiology, Kings College London, St. Thomas Hospital, Westminster Bridge
Road, London SE1 7EH, UK
2
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK

The regulation of gene expression plays a pivotal role in

complex phenotypes, and epigenetic mechanisms such
as DNA methylation are essential to this process. The
availability of next-generation sequencing technologies
allows us to study epigenetic variation at an unprecedented level of resolution. Even so, our understanding of
the underlying sources of epigenetic variability remains
limited. Twin studies have played an essential role in
estimating phenotypic heritability, and these now offer
an opportunity to study epigenetic variation as a dynamic quantitative trait. High monozygotic twin discordance
rates for common diseases suggest that unexplained
environmental or epigenetic factors could be involved.
Recent genome-wide epigenetic studies in disease-discordant monozygotic twins emphasize the power of this
design to successfully identify epigenetic changes associated with complex traits. We describe how large-scale
epigenetic studies of twins can improve our understanding of how genetic, environmental and stochastic factors
impact upon epigenetics, and how such studies can
provide a comprehensive understanding of how epigenetic variation affects complex traits.
Epigenetic mechanisms
The term epigenetics was originally introduced to describe
how interactions between genetics and environment can
give rise to phenotypes during development [1]. Epigenetics today more specifically defines cellular modifications
that can be heritable, but appear unrelated to DNA sequence changes, and can be modified by environmental
stimuli [2,3]. At present, epigenetic mechanisms typically
comprise DNA methylation and histone modifications, but
also include many other mechanisms such as ATP-based
chromatin-remodeling complexes, PolycombTrithorax
protein complexes, non-coding RNA mediated gene-silencing, and potentially prions, transcription-factor binding,
and other mechanisms involved in generating and maintaining heritable chromatin structure and attachment to
the nuclear matrix. Epigenetic mechanisms play an essential functional role in complex organisms as regulators of
transcription. Central to epigenetic regulation is the modulation of chromatin structure, whereby the majority of
epigenetic processes impact upon chromatin organization
and maintenance. Next-generation sequencing technologies have been developed to assay epigenetic changes
Corresponding authors: Bell, J.T. (jordana@well.ox.ac.uk); Spector, T.D.
(tim.spector@kcl.ac.uk).

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(Box 1) in high-throughput approaches, and high-resolution genome-wide epigenetic profiles promise a more complete understanding of the functional impact of
epigenetics. Of these processes, DNA methylation is the
mechanism that has been studied in the greatest depth,
and we therefore focus predominantly on this mechanism
in this review.
Epigenetic mechanisms are present in many taxa, but
DNA methylation has been most extensively studied in
mammals where it appears to be restricted to the cytosine
base, and especially in the context of CpG dinucleotides.
CpG dinucleotides are cytosinephosphateguanine
sequences that typically cluster in genomic regions referred to as CpG islands (CGI), which are often located
in gene promoters and exhibit low levels of DNA methylation. However, DNA methylation in mammals can also
occur outside the CpG context, and this has been reported
for example in embryonic stem cells [4]. Furthermore,
although cytosine is typically methylated to 5-methylcytosine it can also be converted to 5-hydroxymethylcytosine,
which could also play an important epigenetic role [5]. In
mammals, DNA methylation is mediated by DNA methyltransferases that are responsible for de novo methylation
and the maintenance of methylation patterns during replication [6], and also by DNA demethylases that remain
largely unknown. There are several assays for genomewide evaluation of DNA methylation patterns (Box 1), and
methylation cross-technology comparisons have shown
high concordance between different sequence-based methods [7] and slightly lower concordance between sequencedbased and microarray-based methods [8,9].
Cytosine methylation is essential in mammalian development, particularly in cell-lineage specification [1012],
in the regulation of transcription [1318], and in maintaining genome stability [10,16,19]. Correspondingly, variable DNA methylation patterns mirroring the functional
context of genomic regions have been observed in regulatory regions, in promoters and gene-body regions, and in
repetitive elements [10,1923], suggesting that different
mechanisms could be involved in the regulation of DNA
methyltransferase activity across the genome and in the
interaction with chromatin-associated proteins and histone modifications [24,25]. Discrete changes in cytosine
methylation at CpG dinucleotides in gene promoters can
induce stable silencing of gene expression both in normal
development [10] and in disease [26]. Overall, patterns of
negative correlation between promoter methylation and

0168-9525/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2010.12.005 Trends in Genetics, March 2011, Vol. 27, No. 3

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Box 1. Next-generation sequencing technologies applied to epigenetics

The availability of next-generation sequencing technologies has
recently allowed the survey of genome-wide epigenetic variation at
high resolution [8,99,104,105]. We describe some of these approaches
briefly below. In addition, single-molecule sequencing technologies
(e.g. [106]) will probably reveal an even more complex and diverse
layer of epigenetic mechanisms and modifications.

lated cytosines, thereby allowing the separate detection of

methylated cytosines and 5-hydroxymethylated cytosines.
(iii) Methylation-sensitive restriction enzyme sequencing (MRE-seq).
This approach detects typically unmethylated DNA and involves
DNA digestion with methyl-sensitive restriction enzymes, such as
HpaII, Hin6I and AciI, to select unmethylated DNA restriction
enzyme cut-sites, followed by sequencing.

DNA methylation
(i) Bisulfite sequencing (Bi-seq) and reduced-representation Bi-seq
(RRBS). These methods are based on bisulfite conversion of the
DNA followed by sequencing. During bisulfite conversion, unmethylated cytosines are converted to uracils, whereas methylated
cytosines remain unchanged, therefore complete bisulfite conversion is crucial to identifying correctly the unmethylated fraction of
the genome. Bisulfite treatment cannot distinguish between
methylated cytosines and 5-hydroxymethylated cytosines. Bisulfite-sequencing approaches survey DNA methylation across the
entire genome (Bi-seq) or in a fraction of the genome (RRBS),
which tends to be enriched for clusters of CpG dinucleotides.
(ii) Methylated DNA immunoprecipitation sequencing (MeDIP-seq),
methylated DNA capture by affinity purification sequencing
(MeCAP-seq), and methylated DNA binding domain sequencing
(MBD-seq). These techniques are based on extraction of the
methylated sections of the genome by antibody or protein
binding, followed by immunoprecipitation and sequencing. In
some of these technologies the CpG density of a DNA fragment
will affect precipitation, but algorithms that take this into account
have been developed [9]. One advantage of the MeDIP-seq
approach is the option to design antibodies specific to methy-

gene expression have been observed across multiple organisms and tissues [2022,27]. Furthermore, such negative
correlations are more striking in CGI shores, defined as
regions up to 2 kb outside of CGI borders, suggesting a
functional role for these genomic regions in tissue differentiation and disease [27,28]. In addition, in genomic
imprinting only one parent-of-origin copy of the gene is
expressed, and the other is silenced via differential DNA
methylation. For example, differentially methylated
regions (DMRs) at the human H19 locus control imprinting
and gene expression at the maternally imprinted and
transcriptionally-silenced insulin-like growth factor II
(IGF2) locus and at the paternally imprinted and silenced
H19 region [29,30]. DNA methylation is also strongly
correlated with other epigenetic changes, especially histone modifications, implicating shared mechanisms of epigenetic regulation and downstream effects [13,25,31]. The
possibility that transcriptionally silent chromatin could be
a target for de novo DNA methylation has also been
suggested (see [32]). Altogether, multiple factors including
DNA sequence, DNA methylation, histone modifications,
and other epigenetic and transcriptional activity factors
contribute to chromatin regulation, which in turn modulates transcription and affects mammalian development
and disease (Figure 1).
Studies of twins have been crucial to disentangling the
contribution of genetic factors to numerous complex traits.
Twin studies in epigenetics have the potential to address
two important questions. First, to what extent are epigenetic changes heritable and how much variation is there in
epigenetic heritability across the genome? Comparisons
within and between twin-pairs can help to determine the
extent of epigenetic heritability and stability. Second, do

Histone modification
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is
the standard approach used to detect histone-tail modifications.
Non-coding RNAs
Long non-coding RNAs can be detected using RNA-sequencing
studies by selecting RNA transcripts that are not translated and are
absent from protein databases, although the absence of protein
products needs to be experimentally verified. Small non-coding
RNAs, including miRNAs, typically require a separate library protocol
and RNA sequencing experiment.
Chromatin structure assays
Recent technologies have been designed to assay chromatin structure
directly, and include DNase-seq, in which DNase I hypersensitivity
(HS) sites are sequenced [107]; FAIRE-seq, or formaldehyde-assisted
isolation of regulatory elements followed by sequencing, in which
nucleosome-depleted DNA is isolated from human chromatin [108];
sono-seq, where sonication of cross-linked chromatin is followed by
sequencing [109]; 3C-seq, or chromosome conformation capture
sequencing and related approaches [110113]; and nucleosome
positioning assays.

epigenetic factors contribute to complex phenotypes?

Monozygotic (MZ) twins are traditionally regarded as genetically identical, therefore any phenotypic differences
within MZ twin pairs are classically attributed to environmental factors. However, epigenetic variants can also associate with phenotypic differences, and the identification
and interpretation of such associations is currently an
important area of research. Epigenetic studies of disease-discordant MZ twins, who are completely matched
for genetics, age, sex, cohort effects, maternal influences
and common environment, and are closely matched for
other environmental factors, should be considerably more
powerful in detecting disease-related epigenetic differences than epigenetic studies of unrelated disease cases
and controls with different life-histories. In the following
sections we consider the value of twin studies in epigenetics and discuss recent findings highlighting the possibility that epigenetic variation can be transmitted through
generations and impact upon common diseases.
Epigenetic heritability
Heritability is the proportion of the phenotypic variance in
the population that is attributed to genetic variation.
Heritability estimates are traditionally obtained by comparing the extent of similarity between relatives in classical twin studies, twin-adoption studies, sib/half-sib
studies, and transgenerational family studies. Each has
weaknesses, but for most traits twin studies are generally
regarded as the most reliable because they are unbiased by
age effects and offer the ability to separate common environment from genetic effects [33,34]. In twins, heritability
estimates compare concordance rates or intra-class
correlations in monozygotic (MZ) and dizygotic (DZ) twins
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Active transcription

Repressed transcription

Nuclear lamina
700 nm

Chromatin

11 nm

DNA methylation

2 nm

mRNA

Gene-expression

2 nm

Disease status in MZ twin-pairs

Concordant-unaffected

Discordant

Concordant-affected
TRENDS in Genetics

Figure 1. Epigenetic changes and their effects on transcription and disease. Epigenetic variants across multiple levels of chromatin structure, shown here at different levels
of cell resolution in nanometers (nm), associate with gene expression and disease status in a sample of MZ twins. Top, higher-order chromatin loop configurations and
attachment to the nuclear lamina can represent active and repressed chromatin domains that associate with differential gene expression. The next level represents the
chromatin beads on a string configuration, which reflects structural organization into loosely structured (active) and densely packed (repressed) chromatin states. Histone
modifications associated with active transcription (green) and transcription silencing (red) are indicated by colored dots. The next levels of cell resolution depict DNA
methylation (red M) in the promoter regions of the silenced genes and the corresponding differences in gene expression. Bottom, possible effects of these changes on
disease status in a sample of MZ twins, highlighting unaffected-concordant, discordant, and disease-concordant MZ twins.

(Box 2). Twin comparisons of genome-wide epigenetic profiles can determine whether particular regions of the genome have higher epigenetic heritability estimates. In
such regions, DNA methylation would appear to be influenced by genetic variation and DNA methylation variants
would be relatively stable and could associate with genetic
variants.
Several studies have examined DNA methylation patterns in twins. Early work focused on X-chromosome inactivation patterns in females where one X chromosome is
inactivated at random and the silent state of the inactive Xchromosome is maintained by DNA methylation [35]. The
results indicated that skewed X-chromosome inactivation
patterns are more frequent with increasing age and that
underlying heritable patterns are present in this supposedly random process [36]. Subsequent studies focused on
DNA methylation variability and heritability, and its relationship to age. Initial findings, based either on methylation assays at a few genomic regions in a moderate
sample, or on methylation assays at multiple genomic
regions in a small sample, indicated that epigenetic variation at specific genomic regions can be heritable, but can
also change over time. The first large-scale study examined
DNA methylation and histone acetylation at multiple genomic regions in 20 3-year-old and 20 50-year-old Spanish MZ
twin pairs [37] and observed that MZ twins have very
similar epigenetic profiles, indicative of high epigenetic
heritability. However, epigenetic variability increased with
age across multiple tissues and, interestingly, the greatest
differences were observed post hoc in twins who differed
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most in lifestyle. However, the study was cross-sectional

instead of longitudinal, potentially obscuring comparison of
individual variability; furthermore, comparing epigenetic
patterns in fast-growing children and adults might not be
generalizable to the aging process. To this end, a recent
longitudinal study of twin epigenetic heritability assayed
DNA methylation patterns in the promoters of three genes
in 46 MZ and 45 DZ twin pairs sampled at 5 and 10 years of
age [38]. One gene showed evidence for heritability, whereas
methylation differences were present in all genes at an early
age and increased with time. Time-related changes in methylation have been further identified as age-related locusspecific variation in methylation across multiple tissues in
samples of singletons and twins [3941].
Substantive evidence for epigenetic heritability has
been obtained from further studies of age-matched twins
using larger samples [42,43] or higher-resolution DNA
methylation assays [44]. Two locus-specific studies assayed
DNA methylation in the 11p15.5 genomic region surrounding the maternally imprinted insulin-like growth factor II
(IGF2) gene and the paternally imprinted H19 region,
estimating high epigenetic heritability in those regions
using multiple tissues from 182 newborn MZ and DZ twins
[43] and whole blood from 196 adolescent and 176 middleaged MZ and DZ twins [42]. The most recent large-scale
twin study of DNA methylation [44] used a high-resolution
DNA methylation array [45] in three tissues [buccal, gut
and white blood cells (WBC)] in approximately 20 MZ twinpairs and across two tissues (buccal and WBC) in 20 DZ
age-matched twin pairs. Overall, they found that MZ twins

Review
Box 2. Estimating genetic heritability using twin studies
Heritability refers to the proportion of the phenotypic variance in the
population that is attributed to genetic variation, where genetic
variants are represented either by additive genetic values (for
narrow-sense heritability estimates, h2) or total genetic values (for
broad-sense heritability estimates, H2). Twin heritability estimates
usually refer to the narrow-sense heritability (h2), which is the
proportion of the total phenotypic variance in the population that is
attributable to additive genetic effects. Twin estimates of heritability
compare phenotypic similarities in MZ and DZ twins, because MZ
twins are genetically identical and share 100% of their germline
sequence variation, whereas DZ twins are not genetically identical
and share only on average 50% of germline sequence variants. In
addition, MZ and DZ twins share the same uterus and birth date, and
are exposed to very similar environmental factors in early development. In twins, h2 is typically calculated as twice the difference
between MZ and DZ correlations,
h2 2r MZ  r DZ
where rMZ and rDZ are the correlations of the trait in MZ and DZ twins
respectively. The advantage of the classical twin study is that it is
possible to estimate and distinguish between the contributions of
genetic, shared environmental, and unique environmental components to the phenotype. Heritability estimates are population-specific
and, unless environmental factors remain constant, environmentspecific. Interpreting heritability estimates should avoid common
misconceptions [34]; for example, a large estimate of heritability
does not necessarily relate to underlying genetic variants of large
effects, and a small estimate of heritability does not necessarily imply
low additive genetic variance (for in-depth reviews of twin heritability
refer to [33,34]).

have more similar DNA methylation patterns than DZ

twins across tissues. This was greatest in buccal smears
and lowest in WBCs, potentially due to cellular heterogeneity because buccal cells are a homogeneous mixture of
cell types. The most heritable CpG sites were correlated
with functional regions and promoters, suggesting that the
more functionally-relevant methylation signals were under stronger genetic control.
Overall, these findings confirm that DNA methylation is
a heritable trait on a genome-wide basis, but also highlight
the importance of taking age into account when studying
epigenetic processes. Furthermore, the results are consistent with recent population-based findings of quantitative
trait loci (QTL) for DNA methylation [4648], transgenerational and family clustering of methylation patterns
[49,50], and heritable effects of other epigenetic processes
[51,52].
Interpreting epigenetic heritability
DNA methylation patterns can be affected by genetic
variation, environmental changes, heritable and non-heritable changes in other epigenetic processes (for example,
chromatin structure or transcription factor binding might
influence DNA methylation patterns), and stochastic
changes over time. All these factors can contribute to
DNA methylation heritability estimates, which are therefore time-, tissue-, locus- and population-specific. There are
three further important aspects of epigenetic heritability.
First, does twin epigenetic heritability reflect stability in
methylation transmission during mitosis and meiosis?
Second, does epigenetic variation contribute to phenotype
heritability? Finally, how do epigenetic heritability findings relate to time-dependent methylation changes?

Trends in Genetics March 2011, Vol. 27, No. 3

In mammals, maintenance DNA methyltransferases

and histone methyltransferases ensure propagation of
epigenetic marks through mitotic cell divisions with high
fidelity (in the range of 9599% [53,54]) and precision [55].
However, both tissue specificity and meiotic methylation
erasure argue for imperfect stability of methylation transmission. Although twin epigenetic studies are ideal for
estimating the variance due to genetic factors, they could
overestimate the transmissibility of these factors [56].
There is evidence that the rate of transmission of epigenetic marks lessens with each generation [57], suggesting
that epigenetic profiles are likely to be more similar in
families within generations as opposed to between generations (Figure 2). Whereas in plants transgenerational inheritance of DNA methylation can be relatively stable for
up to eight generations [48,50], in animals meiotic epigenetic erasure should obliterate transmission of epigenetic
variants. Although genome-wide data have not yet been
reported, there are a few single-locus examples of transgenerational epigenetic inheritance in animals [58,59]. In
general, twin studies give higher heritability estimates for
common traits than do family studies, particularly in agerelated diseases such as osteoporosis [60]. This could be for
at least two reasons. First, it is possible that a proportion of
epigenetic changes are not faithfully transmitted to offspring during meiosis, and twin epigenetic heritability
overestimates the stability of meiotic methylation transmission. Second, methylation-changes accumulate with
age, and therefore it is possible that age alone could explain
lower family-based epigenetic heritabilities if age is not
adjusted for or if age influences methylation in a non-linear
manner. For example, if DNA was collected from all family
members at birth, then twin-based and family-based epigenetic heritability estimates could be much more similar.
The underlying question is to distinguish between cases
where methylation is completely determined by the genotype and meiotic transmission is very stable, and cases
where meiotic transmission is not stable or genotype does
not affect methylation [55]. Comparisons of epigenetic
heritability estimates from twins and multigenerational
family studies should establish whether twin heritability
directly relates to the stability of meiotic transmission.
Epigenetic twin heritability estimates, transgenerational
studies, and DNA methylation QTL studies suggest that
both scenarios are plausible, but a better understanding of
the mechanisms underlying meiotic transmission, maintenance methylation, and de novo methylation is required.
Epigenetic changes clearly contribute to phenotypes,
but the extent to which they contribute to phenotype
heritability is unknown (Figure 3). Hence, whether epigenetic changes explain part of the missing heritability of
genome-wide association (GWA) studies [61,62] also
remains far from clear. The missing heritability refers to
the paradox that GWA studies have identified many genetic variants associated with complex human diseases
and traits, but most variants explain only a small proportion of familial clustering. To address this point with
respect to epigenetics, a notable recent study [63] extended
the genetic model of Risch [6466] which relates genetic
and phenotypic variation in a mathematical framework.
Slatkin [63] included both epigenetic and genetic factors in
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Time

MZ twins

Epigenetic
similarity

Generation

Past

DZ twins

Epigenetic
similarity

G0

G1

Density

G1

Density

T1

G1 at T1
1

G1 vs G2

G2

G2
1

G2

G3

G3
1

G3

Correlation

G1

Density

G1

Density

G1 vs G3

Correlation

TN

Correlation
Density

Density

T3

G1 vs G2

Correlation
G1 vs G3

Correlation
Density

T2

Density

Correlation

G1 at TN
1

Correlation

Correlation

Present
TRENDS in Genetics

Figure 2. Transgenerational changes in epigenetic variation in twins. The figure provides illustrative examples of epigenetic heritability estimates in hypothetical families
that include pairs of MZ or DZ twins across three generations. This highlights the idea that epigenetic heritability estimates from twin studies are expected to be higher than
those obtained from transgenerational families. At each of three generation (G1, G2, G3) we represent MZ (blue) and DZ (red) intra-class correlation distributions for
genome-wide DNA methylation patterns, and these are consistent with reported correlation estimates [44]. We compare within-generation correlation distributions to
hypothetical transgenerational correlations in DNA methylation from parentoffspring (G1 versus G2) and grandparentgrandchild (G1 versus G3) pairs to illustrate the fact
that epigenetic heritability becomes diluted over generations. In addition, the figure also emphasizes the time-specific aspect of epigenetic heritability estimates and
specifically the reduction in correlation of genome-wide DNA methylation patterns for one pair of twins (at generation G1) with age, where genome-wide methylation
correlations are slightly lower at later (TN) than at earlier (T1) stages in life. This is consistent with the observed increase in epigenetic variance in older twins [37,38].

[()TD$FIG]
DNA
methylation

Complex
phenotypes
0.50
1.00
0.2 [-0.7,1]

1.00
1.00
0.4 [-0.5,1]

Ep
A1 C1

M
One individual

M1

E1

E2 M2

P2

P1
MZ twins

C2

A2

A1 C1

M1

E1

E2 M2

P1

C2

A2

P2
DZ twins
TRENDS in Genetics

Figure 3. Epigenetic and phenotypic heritability. Path diagrams detailing the proposed contribution of latent variables to the methylation status of an individual at one
genomic region (M) and to their phenotype (P). The left panel represents latent variables contributing to DNA methylation status at one genomic region in one individual:
effects will be specific to the age, sex, and population of the individual and the tissue sampled. Methylation latent factors include additive genetic factors (A), common
environmental factors (C), unique environment (E), and heritable and stable epigenetic factors that are not DNA-sequence dependent (Ep). The right panel represents the
path model in twins, depicting the contribution of DNA methylation and other factors to the phenotype (P) in twin i with correlation estimates in MZ (left) and DZ (right)
twins for latent variables including additive genetic effects (Ai), common environment (Ci), DNA methylation (Mi) and unique environment (Ei). Correlation estimates were
obtained from previous genetic [114] and epigenetic studies [44] in twins. In siblings, the correlation in M will probably be lower than that observed in DZ twins due to age
differences and a higher proportion of stochastic changes.

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Review
a single model to estimate their joint contribution to
complex-trait susceptibility. His results suggested that
although epigenetic changes can add to individual disease
risk, they do not necessarily contribute to heritability,
unless the stability of methylation transmission during
meiosis is high [63]. In plants, transgenerational studies
show that transmission of methylation patterns is stable
over up to eight generations [50], but such data are not yet
available for humans. Estimating the epigenetic contribution to phenotype heritability will depend on assumptions
made regarding the stability of methylation transmission
during meiosis, age-, tissue- and population-specific proportions of methylation changes, the contribution of methylation changes to disease risk, and the suitability of the
multiplicative model. More direct data are needed to test
these underlying assumptions and therefore establish
whether epigenetics contributes to the missing heritability. Significant epigenetic contributions to complex phenotypes can also explain the 30-year-old paradox [67] that, in
laboratory bred isogenic mice, artificially created monozygotic twins (from the splitting of a single egg) show a
greater degree of phenotypic similarity than dizygotic
twins (from two fertilized eggs), despite both groups being
apparently identical genetically and housed in controlled
environments [68].
Despite the evidence that DNA methylation is heritable,
substantial changes in methylation patterns can take place
over time [3741,69], suggesting that certain regions of the
genome are either undergoing epigenetic drift, or perhaps
contribute to the aging process. It is therefore important to
obtain estimates for the timing of epigenetic changes and
how long these persist in mitotic transmissions in different
human tissues. Longitudinal studies [38,49,69] imply that
the precise extent of methylation can vary considerably
over the scale of years, however, time-related changes in
methylation tend to be modest at sites that are either
completely methylated or unmethylated. Furthermore,
time-related changes in methylation need to be identified
with respect to disease onset and progression so as to
distinguish between epigenetic changes that could be causal and those that arise secondary to disease. The change in
methylation patterns with age suggests that epigenetic
heritability can be thought of as a dynamic process, whereby a combination of permanent genetic effects reflecting
the identity of the primary DNA sequence, cumulative
stochastic changes occurring at each mitosis, and temporary environmental effects and insults can trigger epigenetic changes, and epigenetic heritability in a specific
genomic region can decrease with time.
The discordant MZ twin model and epigenetics
Phenotype differences between MZ twins reared apart are
not significantly higher than between MZ twins reared
together [70]. Rates of disease discordance in MZ twins are
usually well over 50%, even for highly heritable disease
[7173], suggesting that epigenetics can contribute significantly to MZ twin phenotype discordance [56,74]. Discordant MZ twins have been identified in a number of diseases
with rates of discordance increasing inversely with disease
prevalence, where discordance is calculated as a function of
prevalence. For example, in rheumatoid arthritis (RA) and

Trends in Genetics March 2011, Vol. 27, No. 3

schizophrenia MZ discordance rates are around 80% and

prevalence rates are around 1% [72,75], whereas MZ discordance in osteoarthritis is around 40% and prevalence is
around 20% [76]. Over the past two decades the discordant
MZ twin design has emerged as a powerful tool for detecting phenotype risk factors while controlling for unknown
confounders. Successful discoveries, which had been difficult to achieve from conventional observational epidemiology, include the influence of smoking and alcohol use on
bone, the effects of social class and exercise on aging, and
the causality of C-reactive protein in heart disease and
obesity [7780]. The discordant twin model is therefore
helpful in resolving complex epidemiological questions and
in detecting risks of small individual effect with samples as
small as 2050 twin pairs.
Recently, several epigenetic studies of MZ discordant
twins have examined differences in DNA methylation
profiles, aiming to identify differentially methylated
regions (DMRs) in human disease [8185]. One of the
earliest studies examined methylation in the dopamine
D2 receptor gene (DRD2) in two discordant or concordant
pairs of schizophrenic twins; this study found greater
methylation differences between discordant MZ twins than
in unrelated cases [85]. Two subsequent studies of bipolar
disorder [83] and caudal duplication anomaly [84] also
identified some phenotype-associated methylation changes
in discordant MZ twins, whereas a third study reported
variation in methylation in the catechol-O-methyltransferase gene in birth-weight discordant MZ twins [86]. In
addition to these studies, DNA methylation at single
imprinted regions has also been found to differ between
MZ twins discordant for BeckwithWiedemann syndrome
[87]. All these studies examined relatively small numbers
of twins with relatively low levels of epigenetic coverage. A
recent study of autoimmune disease in 15 pairs of twins
with systemic lupus erythematosus (SLE), RA, and dermatomyositis assayed genome-wide methylation profiles [82].
This study found 49 significant DMRs in SLE involving
immune-system-related genes, but no differences were
observed in RA and dermatomyositis [82]. The most recent
discordant twin study used a genome-wide approach using
RRBS (Box 1) in CD4+ cells from three twin pairs [81] and
reported no clear differences between twins discordant for
multiple sclerosis (MS). However, only three twin pairs
were included in the analysis, and these comprised a
heterogeneous mix of males, females, Europeans and African-Americans. Although these findings could represent
true negative results, or tissue-, age- or disease-heterogeneity, it is also probable that the sample size was too small
to provide statistical power to detect significant epigenetic
differences. This was also the first study to use nextgeneration sequencing technologies to assay methylation
in disease-discordant twins. Further studies should validate these results with larger case numbers and identify a
similar effect in other autoimmune diseases.
The power of discordant MZ twin studies to detect
DMRs will depend on a number of factors, including the
effect size of the epigenetic change on the phenotype, the
similarity of methylation profiles between MZ twins, sample size, and the sensitivity and coverage of the methylation assay. An estimate of the power of the discordant MZ
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twin design for a specific microarray methylation assay
[45] found that a relatively small number (1525) of phenotypically discordant twin pairs had sufficient (>80%)
power to detect epigenetic changes of 1.2-fold, where an
effect size of 1.2-fold change was significantly greater than
the null experimental variance threshold for the assay
(1.15-fold change) [88]. However, these power estimates
do not necessarily apply to other genome-wide methylation
assays, for example MeDIP-seq or Bi-seq (Box 1), which
differ in sensitivity, specificity, and coverage. Therefore,
the issue of power to detect epigenetic changes in the
discordant MZ design needs to be revisited in view of
next-generation technologies.
Future directions in epigenetic studies on twins
There are several aspects of epigenetic studies where twins
present novel opportunities to understand the biology and
the mechanisms underlying complex traits. We suggest a
few examples.
Unraveling phenotypic complexity
Twin resources such as the MuTHER (multiple tissue
human expression resource) project (http://www.muther.ac.uk/), which aims to assay gene expression variation in
multiple tissues in twins and to identify regulatory genetic
variants, can be linked with epigenetic data to explore the
tissue specificity and functional consequences of epigenetic
variation in twins. This project is being extended to RNA
sequencing (via the EUroBATS project), and this will also
allow differential allelic expression to be explored. Allelespecific expression (ASE) patterns are relatively common
and are under strict genetic control [89]. ASE patterns are
of interest because they are often under allele-specific
methylation (ASM) control: they are typically observed
in X-chromosome inactivation and in imprinted regions,
but can also occur in non-imprinted autosomal genes. ASM
is widespread throughout the genome [90,91] and is of
interest in twin studies where a spectrum of ASM is likely
to be observed and could be partitioned into epigenetic
heritability and phenotype contribution. The power of
combining multiple types of biological data in normal
and phenotype-discordant twins will allow us to address
the pleiotropic effects of genetic and epigenetic changes,
that is, changes affecting multiple phenotypes, and help
the interpretation of GWA studies and potentially provide
insight into evolutionary mechanisms.
Are epigenetic changes causal or secondary to the
phenotype?
The timing of the epigenetic changes is crucial to understanding their role in complex traits. There is a need to
measure the baseline epigenetic profile in multiple tissues
before disease onset, ideally at birth or at the beginning of
adulthood, with sampling at regular intervals thereafter.
There are ambitious ongoing efforts to obtain newborntwin epigenetic profiles as part of longitudinal studies
[43,92,93]. DNA methylation analysis of multiple tissues
from newborn twins reveals both genetic and intrauterine
components to variation in the human neonatal epigenome
[43]. These data will also help us understand the effects of
the intrauterine environment on epigenetics.
122

Trends in Genetics March 2011, Vol. 27, No. 3

In vitro fertilization (IVF) twins and epigenetics

Epigenetic profiling at birth in twins is also relevant to
determining whether assisted reproduction technologies
(ARTs) affect epigenetics. ARTs include IVF and related
technologies [94] and have been linked to an increase in
multiple births and low birth weight. About a third of all
European twins are now born as a result of ART. It is
possible that epigenetic marks are introduced as a result
of perturbations in the intrauterine environment associated
with ART, and these could affect early development. Furthermore, birth weight (which has both genetic and environmental influences and is governed by imprinted genes) is
controlled at least in part by epigenetic factors [95,96]. This
could be the mechanism for the Barker or fetal origins
hypothesis that fetal undernutrition in middle-to-late gestation leads to disproportionate fetal growth, and can program later coronary heart disease [97]. However, to date
there is no evidence linking multiple human births following
ART with abnormal epigenetic modifications. To address
the potential role of epigenetics in ART and separate an
inherited (infertility) modification from a secondary one due
to ART, comparisons of epigenetic profiles in non-ART and
ART twins across different ages could be performed [98].
Ongoing large-scale epigenome projects in twins
A recent large-scale study (EpiTwin http://www.twinsuk.
ac.uk/) aims to discover methylated genes responsible for
discordance of ten common traits and diseases. The study
is using MeDIP-seq on blood samples to assay epigenomic
differences in 5000 adult UK twins aged 1685, discordant
and concordant for a wide variety of diseases and environments. Next-generation sequencing, although currently at
significant cost, has the potential to prove powerful in
detecting disease-related methylation differences at a high
level of resolution in a sample of this size. Another ongoing
large-scale prospective study consists of a cohort of Australian newborn twins [43,92]. These data will prove invaluable to unraveling the timing of methylation changes
over the lifetime of an individual. A Norwegian study is
exploring healthy twins for DNA methylation and histonemodification pattern variability across the genome, and
initial findings showed relatively low epigenetic heritability at the major histocompatibility locus [99,100]. Differences in DNA methylation using array-based technologies
are also currently underway in major psychosis [101] and
autism [102]. Lastly, another ongoing project that presents
perhaps a more cost-effective approach to next-generation
epigenomic studies is that undertaken by the ENGAGE
consortium (http://www.euengage.org/), where MeCAP-seq
is being performed by sample pooling across multiple traits
in discordant twins.
Forensics and tissue transplantation
Two further areas that can benefit from the epigenetic
differences observed in MZ twins are forensic science and
medical transplantation. Although twins are no more likely to be criminals than the general population, one in 250
people are MZ twins and legal cases involving MZ twins
are high-profile. For example, the genetic identity of MZ
twins can allow twins to provide each other with alibis in
criminal cases. Differences in epigenetic profiles between

Review
MZ twins, if consistently replicated, could in future lead to
closing this loophole. In transplantation there are reports
of occasional graft failures in identical twins. Studying
subtle differences in twin epigenetic profiles could improve
transplantation outcomes, where small epigenetic changes
of immune-related genes in the host or in transplanted
organs could affect transplant success [103]. Again, having
a baseline of normal epigenetic differences between MZ
twins at different ages could guide the evaluation of epigenetic alterations relevant to transplantation.
Concluding remarks
The study of epigenetic profiles in twins offers an excellent
opportunity to understand the causes and consequences of
epigenetic variation. Twin epigenetic heritability estimates tell us about the genetic control of DNA methylation
variability and the stability of methylation patterns during
cell division. The contribution of epigenetic variants to
complex phenotypes can be assessed using disease-discordant MZ twins who are otherwise matched for genetics,
age, sex, cohort effects, maternal effects and a common
environment. These twin designs are considerably more
powerful discovery tools than studies on singletons. In the
near future, large-scale epigenetic studies in twins across
different ages, tissues, and diseases will improve our understanding of the etiology and mechanisms of a wide
range of common complex traits and diseases.
Acknowledgments
We thank J. Craig, R. Saffery, the anonymous reviewers, and the editor
for their helpful comments. J.T.B. is supported by a Sir Henry Wellcome
postdoctoral fellowship. T.D.S. is a National Institute for Health
Research and European Research Council Senior Investigator and is
funded by the Wellcome Trust.

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