Basic concepts of epigenetics

Michal Inbar-Feigenberg, M.D.,a,b Sanaa Choufani, Ph.D.,a Darci T. Butcher, Ph.D.,a Maian Roifman, M.D.,b
and Rosanna Weksberg, M.D., Ph.D.a,b,c
a
Genetics and Genome Biology and b Division of Clinical and Metabolic Genetics, The Hospital for Sick Children; and
c
Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada

Several types of epigenetic marks facilitate the complex patterning required for normal human development. These epigenetic marks
include DNA methylation at CpG dinucleotides, covalent modications of histone proteins, and noncoding RNAs (ncRNAs). They
function in a highly orchestrated manner, regulating mitotically heritable differences in gene expression potential without altering
the primary DNA sequence. In germ cells and the developing embryo, genome-wide epigenetic reprogramming drives the erasure
and reestablishment of correct epigenetic patterns at critical developmental time periods and in specic cell types. Two specic types
of epigenetic regulation established in early development include X-chromosome inactivation and genomic imprinting; they regulate
gene expression in a dosage-dependent and parent-of-origin-specic manner, respectively. Both genetic and environmental factors
impact epigenetic marks, generating phenotypic variation that ranges from normal variation to human disease. Aberrant epigenetic
patterning can lead to a variety of human disorders, including subfertility and imprinting
disorders. (Fertil Steril 2013;99:60715. 2013 by American Society for Reproductive
Medicine.) Use your smartphone
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A
ll cells in the human body carry developing embryo and fetus. Several females. Genomic imprinting refers to
the same DNA complement, types of epigenetic marks work in a process by which particular genes, car-
which originates from a single concert to drive appropriate gene rying parent-of-origin-specic epige-
cell at conception. Highly orchestrated expression (Fig. 1). These include DNA netic marks, have the potential to drive
epigenetic mechanisms facilitate the methylation at CpG dinucleotides, monoallelic parent-of-origin-specic
complex patterning required to ensure covalent modications of histone gene expression in certain cell types
normal human development and sup- proteins, ncRNAs, and other comple- at specic times in development. In
port stable regulation of appropriate mentary mechanisms controlling germ cells and in the developing
patterns of gene expression in diverse higher order chromatin organization embryo, genome-wide epigenetic
cell types. Epigenetic mechanisms within the cell nucleus. reprogramming underpins the erasure
dene mitotically heritable differences Two unique epigenetic mecha- and reestablishment of correct epige-
in gene expression potential without nisms, X-chromosome inactivation netic patterns. These naturally
altering the primary DNA sequence and genomic imprinting, will be dis- occurring processes are distinct from
(1). These mechanisms are highly regu- cussed as examples of the importance the in vitro reprogramming process
lated by a large number of proteins that of epigenetic regulation in maintaining recently developed to induce pluripo-
establish, read, and erase specic correct patterns of gene expression in tent stem cells from somatic cells (2).
epigenetic modications, thereby early development. X-chromosome While some epigenetic marks are
dening where and when the transcrip- inactivation represents a paradigm for stable over time in particular tissues,
tional machinery can access the dosage compensation in females, others demonstrate developmental
primary DNA sequences to drive nor- resulting in monoallelic expression of plasticity. Epigenetic alterations or
mal growth and differentiation in the large numbers of X-linked genes in epimutations that arise via a number
of different mechanisms can lead to
Received November 21, 2012; revised January 16, 2013; accepted January 17, 2013; published online a variety of human disorders, including
January 26, 2013. subfertility and imprinting disorders.
M.I.-F. has nothing to disclose. S.C. has nothing to disclose. D.T.B. has nothing to disclose. M.R. has
nothing to disclose. R.W. has nothing to disclose. Both genetic and environmental factors
Reprint requests: Rosanna Weksberg, M.D., Ph.D., Division of Clinical and Metabolic Genetics, The impact epigenetic marks, generating
Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada (E-mail:
rweksb@sickkids.ca). phenotypic variation ranging from
normal variation to human disease
Fertility and Sterility Vol. 99, No. 3, March 1, 2013 0015-0282/$36.00 (3). Environmental factors such as
Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.fertnstert.2013.01.117 maternal starvation and the use of

VOL. 99 NO. 3 / MARCH 1, 2013 607

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FIGURE 1
Epigenetic mechanisms affecting gene expression. Epigenetic
patterns are established by a number of mechanisms. Epigenetic
marks include DNA methylation and covalent modications of
histone proteins. DNA methylation is established and maintained by
the DNMT enzymes. DNA is wrapped around histone protein cores
composed of an octamer containing two copies of each core
histone: H2A, H2B, H3, and H4. Together, these form the basic unit
of chromatin, the nucleosome. Histone modications are regulated
by several enzymes including histone acetyltransferases (HATs) and
deacetylases (HDACs). Acetylation of histone proteins by HAT is
commonly found in euchromatin (relaxed state of chromatin) and is
associated with active transcription. Deacetylation of histone
proteins by HDAC and methylation of DNA by DNMTs is a hallmark
of heterochromatin (condensed state of chromatin), which is
associated with transcriptional repression.
Inbar-Feigenberg. Basic concepts of epigenetics. Fertil Steril 2013.
assisted reproductive technologies (ART) have been demon-
strated to impact the epigenome of the embryo. Some of the
epigenetic alterations associated with maternal starvation in
fetal life persist through to adulthood, likely contributing to
late-onset disorders such as cardiovascular disorder and
type 2 diabetes (48).
Epigenetic Marks
DNA methylation, histone modications, and ncRNAs are
described below as independent mechanisms, but it is impor-
tant to note that there is cross-talk between the different
epigenetic marks to regulate the epigenome (9, 10). The
ENCODE Project Consortium, a large collaborative effort
developed to dene all of the functional elements in the
human genome, has recently published large data sets of
transcription, histone modications, and additional protein
binding data. These data annotate both global and regional
overlapping epigenetic features, which in combination
regulate gene expression (11).
DNA Methylation
Currently, one of the best studied epigenetic mechanisms is
DNA methylation (12). DNA methylation is typically associated
with gene silencing through binding of methylation-sensitive
DNA binding proteins and/or by interacting with various
modications of histone proteins that modulate access of
gene promoters to transcriptional machinery (13). In
eukaryotic species, DNA methylation involves transfer of
608
a methyl group to the cytosine of the CpG dinucleotide (13).
The vast majority of mammalian DNA methylation occurs at
CpG dinucleotides (14, 15). CpGs are distributed
nonrandomly in the genome. They are concentrated in
genomic regions called CpG islands ranging in size from 200
bp to several kilobases. These CpG islands, often
unmethylated, are typically located within gene promoters of
actively transcribed housekeeping genes and tumor
suppressor genes (16). In contrast, CpG islands of silent genes
are predominantly methylated (17).
Specic proteins, such as DNA methyltransferases, can
establish or maintain DNA methylation patterns. Studies in
mice demonstrate that DNA methyltransferases are essential
for normal embryonic development (18, 19). DNA
methylation is established de novo by the DNA
methyltranferase (DNMT) enzymes DNMT3a and DNMT3b
and maintained through mitosis primarily by the DNMT1
enzyme (12). Another member of the DNMT3 family,
DNMT3L, has no catalytic activity but can activate
DNMT3A to establish allele-specic methylation in imprinted
regions of the genome (20). DNMT1 is the primary mainte-
nance methyltransferase with a high afnity for hemimethy-
lated DNA (21). Its primary function is to copy the
methylation patterns during replication. DNMT1o, one devel-
opmental stage-specic isoform of DNMT1, is an
oocyte-derived protein that enters nuclei at the eight-cell
stage of early embryos and has an essential role in
maintenance of epigenetic marks (22).
DNA demethylation is also critical during primordial
germ cell (PGC) and early embryo development (23). This
can occur via passive demethylation that is associated with
cell division or via active demethylation using excision repair
mechanisms. The active pathway requires hydroxylation of
the 5-methylcytosine to 5-hydroxymethylcytosine by the
enzymes TET1 and TET2, followed by deamination by AID
and APOBEC1 before base or nucleotide excision repair (23).
All the enzymes in this pathway are expressed in mouse
PGCs, suggesting a role in gametic epigenetic reprogramming
(24, 25).
Histone Modications
The basic unit of chromatin consists of an octamer of histone
proteins, two each of H2A, H2B, H3, and H4. DNA wraps
around this core, which provides structural stability and the
capacity to regulate gene expression (Fig. 1). Each core histone
within the nucleosome contains a globular domain and
a highly dynamic N-terminal tail extending from the globular
domains (Fig. 1). Histone proteins have tails that can have
a number of post-translational modications including
acetylation, methylation, phosphorylation, ubiquitylation,
sumoylation, ADP-ribosylation, proline isomerization, citrul-
lination, butyrylation, propionylation, and glycosylation (26).
Recently published data from the ENCODE Project Consortium
analyzed 11 such histone post-translational modications in-
cluding acetylation and methylation, which mark active and
repressive chromatin, as well as modications associated
with transcription. Assessing various histone modications
in a number of tissues, that data set identied different
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chromatin states including inactive, bimodal, and active, each
of which has different functional properties (27). Bimodal
states, in which a combination of active and repressive marks
are present in the chromatin of a promoter region of a gene,
facilitate rapid changes in gene expression, as might be ex-
pected during early development, when differentiation and
specication occur (23).
Regulatory ncRNAs
ncRNAs are also required for epigenetic regulation of gene
expression. Although eukaryotic genomes transcribe up to
75% of genomic DNA, approximately 3% of these transcripts
encode for proteins; the majority are ncRNAs, which can be
classied according to size and function (11, 27, 28).
Regulatory ncRNAs including small interfering RNAs
(siRNAs), microRNAs (miRNAs), and long ncRNAs
(lncRNAs) play important roles in gene expression
regulation at several levels: transcription, mRNA
degradation, splicing, and translation (29). SiRNAs are
double-stranded RNAs (dsRNA) that mediate
post-transcriptional silencing, in part by inducing hetero-
chromatin to recruit histone deacetylase complexes (30).
MiRNAs comprise a novel class of endogenous, small
(1824 nucleotides in length); single-stranded RNAs gener-
ated from precursor RNA cleaved by two RNA polymerase
III enzymes DROSHA and DICER to produce mature miRNA.
These miRNAs can control gene expression by targeting
specic mRNAs for degradation and/or translational
repression (31, 32). They can also control gene expression
by recruiting chromatin-modifying complexes to DNA
through binding to DNA regulatory regions, thereby altering
chromatin conformation (33, 34). Expression of miRNA in
human blastocysts correlates with maintenance of
pluripotency in embryo development (35).
LincRNAs, a subset of lncRNA, exhibit high conservation
across different species. They have been shown to guide
chromatin-modifying complexes to specic genomic loci,
thereby participating in the establishment of cell typespecic
epigenetic states (36, 37). In embryonic development,
expression of lncRNAs, regulated by the pluripotent
transcription factors OCT4 and NANOG, facilitates cell
lineagespecic gene expression (38). LincRNAs also play
an important role in developmental processes such as
X-chromosome inactivation and genomic imprinting (39).
Specic Types of Epigenetic Regulation: X
Inactivation and Genomic Imprinting
X inactivation and genomic imprinting are specialized forms
of epigenetic regulation essential for correct dosage control of
gene expression for the X-chromosome and for genes with
parent-of-origin-specic expression. The study of these
complex regulatory mechanisms has been important in
elucidating how the entire epigenome is regulated.
X Inactivation
To compensate for gene dosage disparities of X-linked genes,
females silence most of one of their two X-chromosomes
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through a process referred to as X-chromosome inactivation
(40). Embryos containing more than one X-chromosome
(XX, XXX females and XXY males) undergo random
X-chromosome inactivation at the blastocyst stage in early
embryogenesis (41). X-chromosome inactivation is regulated
by a master switch locus, the X inactivation center (XIC),
which regulates in cis the expression of the lincRNA gene
XIST (X-inactive specic transcript) and its antisense
transcription unit TSIX. In mouse, the XIC senses the number
of X-chromosomes in the cell and randomly silences all but
one of the X-chromosomes. This involves coating of all future
inactive X-chromosomes by Xist RNA, followed by polycomb
repressor complex 2 recruitment and the addition of silencing
chromatin marks such as histone H3 and H4 hypoacetylation,
H3 lysine27 methylation, and DNA methylation at CpG-rich
promoters (42, 43). This process, the regulation of which is
still incompletely understood, restricts each diploid cell to
one active copy of the X-chromosome.
Genomic Imprinting
Genomic imprinting is an epigenetic process by which the
male and female germ lines confer specic marks or
imprints onto certain chromosomal regions (44).
These imprints provide the potential for monoallelic
parent-of-origin-specic expression at certain times in
development or in specic cell types.
Normal imprinted gene expression is dependent on
a normal relative number of specic parental alleles and
requires contributions from the alleles of both parents. That
is, changing the number or proportion of parental alleles leads
to imprint deregulation. The most extreme example of change
in the relative parental contributions occurs in conceptuses
that contain only maternal or only paternal genomic contri-
butions. Only maternally derived chromosomes are found in
mature cystic ovarian teratomas (MCT), which arise from
a meiotic error during oocyte maturation (gynogenetic
conceptus). The result is the formation of a cyst containing
tissues from each of the three embryonic germ cell layers. In
contrast, an androgenetic conceptus, carrying two paternal
genomes and no maternal genome in all cells, fails to drive
embryonic/fetal development and results in a hydatidiform
mole (AnCHM). MCT and AnCHM display the serious
biological consequences of uniparental inheritance (45), dem-
onstrating that both parental genomes are required for
normal development of an embryo, with the paternal genome
being required for extraembryonic tissues and the maternal
genome being required for embryonic development.
Most autosomal genes are normally expressed from both
paternal and maternal alleles, whereas imprinted genes are
expressed predominantly, or exclusively, from either the
maternal or paternal allele in a parent-of-origin-specic
manner. That is, an imprinted gene is expressed from the
paternal allele while the maternal copy is silenced, or,
conversely, it is expressed from the maternal allele while
the paternal allele is silenced (46). Imprinted genes do not
conform to Mendelian genetics and do not predict
parent-of-origin specicity for gene expression (47). About
100 human genes have been reported to be imprinted
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(http://igc.otago.ac.nz/home.html). Many such genes play
vital roles in embryonic, fetal, and placental growth, as well
as in neurodevelopment (4749).
Imprinted genes tend to cluster together to form
imprinted domains. In humans, imprinted domains have
been found on chromosomes 6, 7, 11, 14, 15, and 20
(4951). Within these domains, imprinted genes are
regulated by imprinting centers (ICs). ICs are characterized
by the presence of differentially methylated regions (DMRs).
Such DMRs carry parent-of-origin-specic DNA methylation
and histone modication marks. These cis-acting DMRs,
along with trans-acting factors, form the basis of the
parent-of-origin-specic gene expression of imprinted genes.
For example, the insulin growth factor2 (IGF2/H19) IC core-
gulates the paternal expression of IGF2 with the maternal
expression of H19, two genes located in the same imprinted
domain 90 kb apart (52).
Epigenetic Reprogramming in Mammalian
Development
Our current knowledge about how the epigenome is reprog-
rammed in early human development comes largely from
extensive research on mouse embryos. Genome-wide epigenetic
reprogramming takes place at two pivotal developmental stages:
during gametogenesis and during early embryogenesis. The rst
event occurs in the PGCs, where epigenetic marks, both
tissue-specic and parent-of-origin-specic imprints, are erased.
New epigenetic marks, including parent-of-origin-specic
imprints, are established at specic developmental time points
both before and after fertilization. After fertilization, a second
wave of reprogramming of epigenetic marks occurs in the early
embryo, including global demethylation followed by de novo
DNA methylation to allow for totipotency and subsequent
cell-specic differentiation and lineage commitment (Fig. 2).
Notably, epigenetic marks at ICs are protected from this wave
of genome-wide epigenetic reprogramming.
Epigenetic Reprogramming in PGCs
Genome-wide erasure and reprogramming of epigenetic marks
is initiated in the PGCs. The sex of the developing embryo
dictates which gamete, the egg or the sperm, will develop
from such early precursors. This sex-specic differentiation
includes the establishment of parent-of-origin-specic
imprints in the sperm and oocytes.
Studies in mice have shown that PGCs begin their
migration toward the developing genital ridge at day 8.5 of
embryonic life in mouse (E8.5), reaching their end point by
E11.5 (53). Genome-wide demethylation of the PGCs is
thought to occur mainly between E11.5 and E13.5, at the
site of the future gonads (54). The decrease in methylation
is global; only 7% of CpGs remain methylated compared
with 70%80% in embryonic stem cells (ES) and somatic cells
(25). Germ cellspecic gene promoters are methylated in
early PGCs, become demethylated, and are expressed during
reprogramming (55). At this time as well, PGCs of female
embryos down-regulate Xist RNA expression from the
inactive X-chromosome (Xi) (56, 57) so that two equivalent
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FIGURE 2
DNA methylation reprogramming in preimplantation embryos. Soon
after fertilization both paternal (blue) and maternal (pink) pronuclei
undergo genome-wide demethylation. Although demethylation of
the maternal genome lags behind the paternal genome, both are
remethylated around the time of implantation (yellow arrow). In
contrast to most other genes, epigenetic marks, such as DNA
methylation, at imprinted loci (ICs) are established early in germ line
development and are protected from the global wave of
demethylation early in embryonic development by maintenance
DNMT isoforms Dnmt1o, Dnmt1, and several other genes such as
NLPR2, NLPR7, and Zfp57. The epigenetic reprogramming of the
preimplantation embryo also includes X inactivation. Notably, ART
takes place during developmental time periods that involve
epigenetic reprogramming. Adapted from reference (109).
Inbar-Feigenberg. Basic concepts of epigenetics. Fertil Steril 2013.
X-chromosomes may participate in meiosis for the
production of female gametes.
De novo methylation in PGCs is established by recruitment
of Dnmt3a and Dnmt3l (58). The KRAB zinc nger protein
ZFP57 also appears to play an important role specically in oo-
cyte imprint establishment (50, 59). Interestingly, the timing of
gamete development differs between male and female embryos.
Spermatogenesis occurs prenatally, and the associated
paternal-specic DNA methylation programming is complete
by birth (60). The epigenetic reprogramming of oocytes occurs
much later than in sperm. It begins at puberty and is nearly
complete in each oocyte at the time of ovulation (60).
Developmental Epigenetic Reprogramming in the
Early Embryo
Before fertilization, the sperm and the egg are highly special-
ized, differing in their gene expression as dictated by distinct
patterns of DNA methylation and chromatin organization
(61). DNA in the sperm is tightly condensed by protamines.
Early embryo reprogramming begins with paternal genome
decondensing as these protamines are replaced by mater-
nally derived histones (61). Soon after fertilization, the
paternal pronuclear genome undergoes rapid demethylation
(Fig. 2) (6268). Demethylation in the maternal genome
follows, such that at the 4-cell stage of the embryo,
the DNA methylation status of the two parental genomes
are equalized (68). Epigenetic marks at imprinted genes are
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protected by specic proteins (Fig. 2) from epigenetic reprog-
ramming throughout the preimplantation development of the
embryo (54).
Lineage Commitment
Lineage commitment is well underway in the blastocyst, with
the formation of the inner cell mass (ICM) and trophectoderm
(TE), each carrying a unique epigenetic signature (61).
Declining levels of the 5-hydroxymethylcytosine (5hmC)
and TET oxidases accompany progressive differentiation
(69, 70). As a result, 5-methylcytosine reaccumulates, possibly
silencing the genes responsible for early developmental regu-
lation (69, 70). By the time cells become fully committed to
their lineage, they will have attained a distinct epigenetic
signature reecting their phenotype, developmental history,
and environmental inuences (61). Interestingly, placental
cells, derived from the trophectoderm, remain relatively
hypomethylated, compared with the differentiated deriva-
tives of the ICM (71), in keeping with the role of the
placenta in endometrial invasion as well as its limited need
for extensive differentiation and longevity (61).
Epigenetic Mark Maintenance
Once established, methylation and other epigenetic marks in
both somatic and germ line cells must be maintained over
the course of future cell divisions. Several genes have been
found to play a major role in this maintenance process in
addition to DNMT1 and DNMT1o; these include NLPR2,
NLPR7, and ZFP57 (59, 7275). As mentioned earlier, the
ZFP57 protein functions in establishing new imprints in
oocytes; it is also needed for maintenance of methylation
more globally (59).
Epigenetic Deregulation and Human Disease
Epigenetic deregulation can result from disturbances in epige-
netic control mechanisms or from alterations in the epigenetic
marks themselves, either in imprinted or nonimprinted genes.
The development of multiple organ systems can be disrupted
TABLE 1
Genetic and epigenetic alterations in BWS.
Genetic
alterations Sequence changes
Mutation of the
maternalCDKN1C
allele chromosome
11p15.5
Epigenetic
alterations
Loss of methylation at IC2
on the maternal chromosome
Combined
alteration
Inbar-Feigenberg. Basic concepts of epigenetics. Fertil Steril 2013.
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by deregulation of epigenetic control mechanisms. Mutations
in proteins that establish, erase, or read epigenetic marks can
negatively affect the development of multiple organ systems,
as can be seen in a variety of human genetic syndromes
such as ATRX (OMIM#301040; alpha thalassemia, mental re-
tardation) (12), Rubinstein-Taybi (OMIM#180849; CREBBP/
EP300) (76), CHARGE (OMIM#214800; CHD7) (77), and Rett
syndromes (OMIM#312750; MECP2) (78).
An epigenetic alteration or epimutation refers to an
aberrant DNA methylation or histone modication pattern.
Such alterations occur in the absence or presence of an
underlying genomic alteration and are called primary and
secondary epimutations, respectively. A primary epimutation
can result from aberrant erasure, establishment, or mainte-
nance of epigenetic marks (50).
In nonimprinted genes, somatic epimutations caused by
mitotic errors in the normal maintenance of epigenetic marks
can result in abnormal growth regulation. For example,
approximately 20% of sporadic breast cancer displays hyper-
methylation of the BRCA1 promoter, often in combination
with an inherited or sporadic mutation on the second allele
(79). Reduced growth potential can also result from an
epimutation. For example, promoter methylation of WNT2
(Wingless-type MMTV integration site family, member 2) in
placenta is associated with reduced birth weight percentile
in the neonate (80), demonstrating that a single epigenetic
hit may impact human growth phenotypes.
Imprinting and Human Disease
Imprinted genes are subject to more complex epigenetic
regulation than nonimprinted genes, providing more oppor-
tunities to acquire epigenetic errors. The relative parental
contributions of specic imprinted regions across the genome
can be disrupted by a number of different mechanisms,
including genetic and/or epigenetic alterations (Table 1).
Primary epimutations (i.e., those without associated
genomic alterations) can arise from stochastic errors in
imprint reprogramming. For example, failure to erase imprints
will result in a gamete of one sex carrying the imprints of the
other sex. When this gamete proceeds to fertilization, the
Cytogenetic abnormalities
Mutations in Submicroscopic genomic Duplication, inversion, or
NLRP2 at 19q13.42 alteration (duplication/deletion) translocation of 11p15.5
within chromosome 11p15.5
Methylation abnormalities
Gain of methylation at IC1 on the Methylation alterations at multiple
maternal chromosome imprinted loci
Parent of origin changes of gene copies
Paternal UPD of 11p15
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resulting zygote will carry uniparental imprints on both
parental chromosomes. Failure to establish appropriate
imprints in the sperm or oocyte may lead to various disorders
of growth and development, including subfertility (81).
Genomic alterations that contribute to imprinting disorders
include deletions and duplications of imprinted regions. Also as-
sociated with imprinting disorders is uniparental disomy (UPD),
in which both homologues of a chromosomal region/segment
are inherited from only one parent (46, 82), that is, there are
two copies of an imprinted region from one parent and none
from the other. UPD of nonimprinted genomic regions is
generally not associated with disease, whereas UPD at specic
genomic locations containing imprinted regions can result in
imprinting disorders. The extent of UPD may range from
a small genomic segment to the entire chromosome (83). The
incidence of UPD of any chromosome is estimated to be about
1:3,500 live births (84). Since deletion or duplication of an
imprinted genomic region changes the relative parental
contributions, it represents another mechanism by which
imprinted genes can be regulated.
Many imprinted genes are expressed in the placenta and
also function in fetal growth regulation and brain
development (44). Epigenetic and associated genetic
aberrations at imprinted genes result in human diseases that
often reect aberrant growth and/or aberrant neurodevelop-
ment. As outlined below, it is very common for individual
imprinting disorders to demonstrate etiologic heterogeneity.
Further, different molecular etiologies may demonstrate
epigenotype/genotype-phenotype correlations.
Neurologic and psychiatric disorders in which deregulation
of imprinted genes are involved and parent-of-origin effects are
observed (85) include Prader-Willi syndrome (PWS) and
Angelman syndrome (AS), two distinct neurodevelopmental
syndromes that both map to the imprinted gene cluster on
chromosome 15q11-q13 (86). These syndromes serve as
an important example of how parental origin of the
genomic/epigenomic lesion denes which imprinting disorder
occurs. A paternal deletion of chromosome 15q11-13 or maternal
UPD15q11-13 leads to PWS, whereas a maternal deletion of chro-
mosome 15q11-13 or paternal UPD15q11-13 leads to AS (50).
One of the best known pediatric growth disorders caused
by abnormal imprinting is Beckwith-Wiedemann syndrome
(BWS). BWS is characterized by macrosomia, macroglossia,
and visceromegaly; embryonal tumors such as Wilms,
hepatoblastoma, neuroblastoma, and rhabdomyosarcoma;
omphalocele, neonatal hypoglycemia, ear creases/pits,
adrenocortical cytomegaly, and renal abnormalities (46).
Pregnancies in which the fetus has BWS may be complicated
by polyhydramnios, enlarged placenta, a long thickened
umbilical cord, increased risk of premature delivery (87),
and placental mesenchymal dysplasia (88). The molecular
etiology of BWS includes two different primary epimutations:
hypomethylation of the proximal IC (IC2) on the maternal
chromosome and hypermethylation at the distal IC (IC1) on
the maternal chromosome. Hypermethylation at IC2 can
also occur as a secondary epimutation when there is an
underlying deletion of the maternal IC2 region. In cases of
BWS with paternal UPD of chromosome 11p15.5, genomic
imprints at both IC1 and IC2 are affected (51). Patients with
612
BWS demonstrate important epigenotype-phenotype
correlations. For example, the highest tumor risks occur in
BWS cases with molecular abnormalities that include
deregulation of IC1.
Russell-Silver syndrome (RSS) is characterized by poor
pre- and postnatal growth, dysmorphic features, and variable
presence of limb/body asymmetry and developmental delay.
To date, two different epigenetic defects have been associated
with RSS (89). The rst is maternal UPD of chromosome 7
occurring in about 10% of RSS cases (90).
The second is hypomethylation at the IC1 on the paternal
chromosome 11p15.5, which occurs in 45% of cases (89,
9193). The latter form is essentially opposite, both in
phenotype and epigenotype, to cases of BWS with gain of
methylation of the same region. Interestingly, some men
with oligospermia have been found to lack paternal
methylation at the IGF2/H19 IC1 on chromosome 11p15.5
in their sperm (94). Children who inherit this chromosome
from their fathers are at risk for developing RSS (89).
Imprinting disorders can include imprinting errors at
multiple imprinted domains. For example, in both BWS and
RSS, some patients exhibit loss of methylation not only at
the IC on chromosome 11p15-IC1 for RSS and IC2 for BWS
but also at other imprinted loci. The molecular basis of this
multilocus loss of methylation (MLOM) is not known for these
two conditions. However, MLOM also occurs in the imprint-
ing disorder transient neonatal diabetes mellitus (74), where
the primary imprinting defect occurs at PLAGL1 DMR on
chromosome 6q24; associated LOM at other maternal DMRs
have been reported (74, 75). The cause of this MLOM
condition has been shown to be homozygous mutations in
the ZFP57 gene. This gene normally encodes an oocyte-
derived maternal factor that participates in preimplantation
maintenance of imprints at multiple loci.
Mutations in NLRP genes can also be associated with
imprinting errors at multiple genomic loci. NLRP proteins are
members of the CATERPILLER protein family that is involved
in inammation and apoptosis (50). Mutations of NLRP7 in the
oocyte are associated with aberrant imprints at multiple loci
(72), causing formation of partial hydatidiform mole, which
is termed biparental due to its oocyte and sperm origins.
Epigenetic Deregulation in Fetal Development
Early embryonic development has been shown to be a critical
period of susceptibility to epigenetic deregulation. Indeed,
studies of children born after maternal starvation have shown
that it is the rst trimester of pregnancy that is a critical period
for epigenetic changes (3, 5, 94). Interestingly, children born
after the use of ART have also been identied to have
changes in epigenetic modications, which are described
below (95, 96).
ART
ART accounts for 1%2% of live births in developed countries
(95). In general, such technologies are highly successful, with
few reports of adverse outcomes. An increased incidence of
epigenetic errors leading to imprinting disorders such as
VOL. 99 NO. 3 / MARCH 1, 2013

BWS, RSS (96), and AS (95) is reported in some studies of
children conceived by ART. However, not all studies have
supported such associations (97). The variability in the
outcomes of such studies is likely due to the large numbers
of variable factors that could affect epigenetic reprogram-
ming. It has been shown that subfertility itself could be
associated with epigenetic errors in the sperm of oligospermic
males (81). Data from mice shows that epigenetic errors can
also arise from ovulation induction or in vitro culture of
embryos (98). We expect that future investigations of new re-
productive technologies will elucidate specic risk factors as-
sociated with ART. In addition, we expect that only some
individuals who undertake ART will be genetically susceptible
to such environmental risk factors.
Environmental Effects
Environmental factors can alter primary epigenetic marks, im-
pacting not only the developing embryo but also the next gen-
eration of offspring. Delineating such transgenerational
environmental effects will be an important focus of future
research. A documented example of the importance of
environment on the evolution of epigenetically based disease
arises from the Dutch and Chinese famine studies (3, 5, 94).
For example, male offspring who were exposed to the Dutch
famine of 194445 within the rst 10 weeks after conception
demonstrated persistent methylation changes at the imprinted
IGF2 gene six decades later (6, 100). The historical famines
provide powerful natural experiments in humans that
resulted in the discovery of epigenetic marks that may be
modied by the prenatal environment (99). Prenatal exposure
to the famine was associated with various adverse metabolic
and mental phenotypes later in life, including a higher body
mass index (100102), elevated plasma lipids (103), increased
risk of schizophrenia (100102, 104, 105), and possible
cardiovascular disease (106).
Exposure to nutritional factors, environmental pollut-
ants, and medications during pregnancy may also alter fetal
epigenetic marks. For example, choline intake during preg-
nancy was found to increase placental promoter methylation
of the cortisol regulation genes CRH and NR31C, leading to
improved stress response in children by lowering cortisol
levels via the hypothalamic-pituitary-adrenal axis (107).
Exposure to toxicants such as the pesticide vinclozolin,
plastics, dioxin, or jet fuel were found to affect fetal and
postnatal development in rats, resulting in changes consistent
with the biology of primary ovarian insufciency and
polycystic ovarian syndrome in humans (108). Some of
these epigenetic changes were transmitted to the next
generation (109).
The role of epigenetic marks in translating the primary
genomic sequence has now moved to the forefront of human
genetic studies. Current epigenetic research is addressing
a number of topics that will add to our understanding of the
role of epigenetic mechanisms in human health and disease,
including characterization of cis- and trans-acting inuences
on the genetic background; delineating cell and tissue-
specic epigenetic marks; determining the interactions
between epigenome and environment, with a focus on fetal
VOL. 99 NO. 3 / MARCH 1, 2013
Fertility and Sterility
programming; uncovering the effects of medication and
nutrition; and assessing risks for adult-onset disorders.
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