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; andc 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 marksinclude DNA methylation at CpG dinucleotides, covalent modifications of histone proteins, and noncoding RNAs (ncRNAs). Theyfunction in a highly orchestrated manner, regulating mitotically heritable differences in gene expression potential without alteringthe primary DNA sequence. In germ cells and the developing embryo, genome-wide epigenetic reprogramming drives the erasureand reestablishment of correct epigenetic patterns at critical developmental time periods and in specific cell types. Two specific typesof epigenetic regulation established in early development include X-chromosome inactivation and genomic imprinting; they regulategene expression in a dosage-dependent and parent-of-origin-specific manner, respectively. Both genetic and environmental factorsimpact epigenetic marks, generating phenotypic variation that ranges from normal variation to human disease. Aberrant epigenetic

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patterning can lead to a variety of human disorders, including subfertility and imprintingdisorders. (Fertil Steril� 2013;99:607–15. �2013 by American Society for ReproductiveMedicine.)Key Words: Epigenetics, DNA methylation, histone modification, microRNA, epigeneticreprogramming, X inactivation, genomic imprinting

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A ll cells in the human body carrythe same DNA complement,which originates from a single

cell at conception. Highly orchestratedepigenetic mechanisms facilitate thecomplex patterning required to ensurenormal human development and sup-port stable regulation of appropriatepatterns of gene expression in diversecell types. Epigenetic mechanismsdefine mitotically heritable differencesin gene expression potential withoutaltering the primary DNA sequence(1). These mechanisms are highly regu-lated by a large number of proteins thatestablish, read, and erase specificepigenetic modifications, therebydefining where and when the transcrip-tional machinery can access theprimary DNA sequences to drive nor-mal growth and differentiation in the

Received November 21, 2012; revised January 16, 20January 26, 2013.

M.I.-F. has nothing to disclose. S.C. has nothing to dnothing to disclose. R.W. has nothing to disclos

Reprint requests: Rosanna Weksberg, M.D., Ph.D., DHospital for Sick Children, 555 University Avenrweksb@sickkids.ca).

Fertility and Sterility® Vol. 99, No. 3, March 1, 2013Copyright ©2013 American Society for Reproductivehttp://dx.doi.org/10.1016/j.fertnstert.2013.01.117

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developing embryo and fetus. Severaltypes of epigenetic marks work inconcert to drive appropriate geneexpression (Fig. 1). These include DNAmethylation at CpG dinucleotides,covalent modifications of histoneproteins, ncRNAs, and other comple-mentary mechanisms controllinghigher order chromatin organizationwithin the cell nucleus.

Two unique epigenetic mecha-nisms, X-chromosome inactivationand genomic imprinting, will be dis-cussed as examples of the importanceof epigenetic regulation in maintainingcorrect patterns of gene expression inearly development. X-chromosomeinactivation represents a paradigm fordosage compensation in females,resulting in monoallelic expression oflarge numbers of X-linked genes in

13; accepted January 17, 2013; published online

isclose. D.T.B. has nothing to disclose. M.R. hase.ivision of Clinical and Metabolic Genetics, Theue, Toronto, Ontario M5G 1X8, Canada (E-mail:

0015-0282/$36.00Medicine, Published by Elsevier Inc.

females. Genomic imprinting refers toa process bywhichparticular genes, car-rying parent-of-origin-specific epige-netic marks, have the potential to drivemonoallelic parent-of-origin-specificgene expression in certain cell typesat specific times in development. Ingerm cells and in the developingembryo, genome-wide epigeneticreprogramming underpins the erasureand reestablishment of correct epige-netic patterns. These naturallyoccurring processes are distinct fromthe in vitro ‘‘reprogramming’’ processrecently developed to induce pluripo-tent stem cells from somatic cells (2).

While some epigenetic marks arestable over time in particular tissues,others demonstrate developmentalplasticity. Epigenetic alterations or‘‘epimutations’’ that arise via a numberof different mechanisms can lead toa variety of human disorders, includingsubfertility and imprinting disorders.Both genetic and environmental factorsimpact epigenetic marks, generatingphenotypic variation ranging from‘‘normal variation’’ to human disease(3). Environmental factors such asmaternal starvation and the use of

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FIGURE 1

Epigenetic mechanisms affecting gene expression. Epigeneticpatterns are established by a number of mechanisms. Epigeneticmarks include DNA methylation and covalent modifications ofhistone proteins. DNA methylation is established and maintained bythe DNMT enzymes. DNA is wrapped around histone protein corescomposed of an octamer containing two copies of each corehistone: H2A, H2B, H3, and H4. Together, these form the basic unitof chromatin, the nucleosome. Histone modifications are regulatedby several enzymes including histone acetyltransferases (HATs) anddeacetylases (HDACs). Acetylation of histone proteins by HAT iscommonly found in euchromatin (relaxed state of chromatin) and isassociated with active transcription. Deacetylation of histoneproteins by HDAC and methylation of DNA by DNMTs is a hallmarkof heterochromatin (condensed state of chromatin), which isassociated with transcriptional repression.Inbar-Feigenberg. Basic concepts of epigenetics. Fertil Steril 2013.

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assisted reproductive technologies (ART) have been demon-strated to impact the epigenome of the embryo. Some of theepigenetic alterations associated with maternal starvation infetal life persist through to adulthood, likely contributing tolate-onset disorders such as cardiovascular disorder andtype 2 diabetes (4–8).

Epigenetic Marks

DNA methylation, histone modifications, and ncRNAs aredescribed below as independent mechanisms, but it is impor-tant to note that there is cross-talk between the differentepigenetic marks to regulate the epigenome (9, 10). TheENCODE Project Consortium, a large collaborative effortdeveloped to define all of the functional elements in thehuman genome, has recently published large data sets oftranscription, histone modifications, and additional proteinbinding data. These data annotate both global and regionaloverlapping epigenetic features, which in combinationregulate gene expression (11).

DNA Methylation

Currently, one of the best studied epigenetic mechanisms isDNAmethylation (12). DNAmethylation is typically associatedwith gene silencing through binding of methylation-sensitiveDNA binding proteins and/or by interacting with variousmodifications of histone proteins that modulate access ofgene promoters to transcriptional machinery (13). Ineukaryotic species, DNA methylation involves transfer of

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a methyl group to the cytosine of the CpG dinucleotide (13).The vast majority of mammalian DNA methylation occurs atCpG dinucleotides (14, 15). CpGs are distributednonrandomly in the genome. They are concentrated ingenomic regions called CpG islands ranging in size from 200bp to several kilobases. These CpG islands, oftenunmethylated, are typically located within gene promoters ofactively transcribed housekeeping genes and tumorsuppressor genes (16). In contrast, CpG islands of silent genesare predominantly methylated (17).

Specific proteins, such as DNA methyltransferases, canestablish or maintain DNA methylation patterns. Studies inmice demonstrate that DNA methyltransferases are essentialfor normal embryonic development (18, 19). DNAmethylation is established de novo by the DNAmethyltranferase (DNMT) enzymes DNMT3a and DNMT3band maintained through mitosis primarily by the DNMT1enzyme (12). Another member of the DNMT3 family,DNMT3L, has no catalytic activity but can activateDNMT3A to establish allele-specific methylation in imprintedregions of the genome (20). DNMT1 is the primary mainte-nance methyltransferase with a high affinity for hemimethy-lated DNA (21). Its primary function is to copy themethylation patterns during replication. DNMT1o, one devel-opmental stage-specific isoform of DNMT1, is anoocyte-derived protein that enters nuclei at the eight-cellstage of early embryos and has an essential role inmaintenance of epigenetic marks (22).

DNA demethylation is also critical during primordialgerm cell (PGC) and early embryo development (23). Thiscan occur via passive demethylation that is associated withcell division or via active demethylation using excision repairmechanisms. The active pathway requires hydroxylation ofthe 5-methylcytosine to 5-hydroxymethylcytosine by theenzymes TET1 and TET2, followed by deamination by AIDand APOBEC1 before base or nucleotide excision repair (23).All the enzymes in this pathway are expressed in mousePGCs, suggesting a role in gametic epigenetic reprogramming(24, 25).

Histone Modifications

The basic unit of chromatin consists of an octamer of histoneproteins, two each of H2A, H2B, H3, and H4. DNA wrapsaround this core, which provides structural stability and thecapacity to regulate gene expression (Fig. 1). Each core histonewithin the nucleosome contains a globular domain anda highly dynamic N-terminal tail extending from the globulardomains (Fig. 1). Histone proteins have tails that can havea number of post-translational modifications includingacetylation, methylation, phosphorylation, ubiquitylation,sumoylation, ADP-ribosylation, proline isomerization, citrul-lination, butyrylation, propionylation, and glycosylation (26).Recently published data from the ENCODE Project Consortiumanalyzed 11 such histone post-translational modifications in-cluding acetylation and methylation, which mark active andrepressive chromatin, as well as modifications associatedwith transcription. Assessing various histone modificationsin a number of tissues, that data set identified different

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chromatin states including inactive, bimodal, and active, eachof which has different functional properties (27). Bimodalstates, in which a combination of active and repressive marksare 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 andspecification occur (23).

Regulatory ncRNAs

ncRNAs are also required for epigenetic regulation of geneexpression. Although eukaryotic genomes transcribe up to75% of genomic DNA, approximately 3% of these transcriptsencode for proteins; the majority are ncRNAs, which can beclassified 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 expressionregulation at several levels: transcription, mRNAdegradation, splicing, and translation (29). SiRNAs aredouble-stranded RNAs (dsRNA) that mediatepost-transcriptional silencing, in part by inducing hetero-chromatin to recruit histone deacetylase complexes (30).

MiRNAs comprise a novel class of endogenous, small(18–24 nucleotides in length); single-stranded RNAs gener-ated from precursor RNA cleaved by two RNA polymeraseIII enzymes DROSHA and DICER to produce mature miRNA.These miRNAs can control gene expression by targetingspecific mRNAs for degradation and/or translationalrepression (31, 32). They can also control gene expressionby recruiting chromatin-modifying complexes to DNAthrough binding to DNA regulatory regions, thereby alteringchromatin conformation (33, 34). Expression of miRNA inhuman blastocysts correlates with maintenance ofpluripotency in embryo development (35).

LincRNAs, a subset of lncRNA, exhibit high conservationacross different species. They have been shown to guidechromatin-modifying complexes to specific genomic loci,thereby participating in the establishment of cell type–specificepigenetic states (36, 37). In embryonic development,expression of lncRNAs, regulated by the pluripotenttranscription factors OCT4 and NANOG, facilitates celllineage–specific gene expression (38). LincRNAs also playan important role in developmental processes such asX-chromosome inactivation and genomic imprinting (39).

Specific Types of Epigenetic Regulation: XInactivation and Genomic Imprinting

X inactivation and genomic imprinting are specialized formsof epigenetic regulation essential for correct dosage control ofgene expression for the X-chromosome and for genes withparent-of-origin-specific expression. The study of thesecomplex regulatory mechanisms has been important inelucidating 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 randomX-chromosome inactivation at the blastocyst stage in earlyembryogenesis (41). X-chromosome inactivation is regulatedby a master switch locus, the X inactivation center (XIC),which regulates in cis the expression of the lincRNA geneXIST (X-inactive specific transcript) and its antisensetranscription unit TSIX. In mouse, the XIC senses the numberof X-chromosomes in the cell and randomly silences all butone of the X-chromosomes. This involves coating of all futureinactive X-chromosomes by Xist RNA, followed by polycombrepressor complex 2 recruitment and the addition of silencingchromatin marks such as histone H3 and H4 hypoacetylation,H3 lysine27 methylation, and DNA methylation at CpG-richpromoters (42, 43). This process, the regulation of which isstill incompletely understood, restricts each diploid cell toone active copy of the X-chromosome.

Genomic Imprinting

Genomic imprinting is an epigenetic process by which themale and female germ lines confer specific marks or‘‘imprints’’ onto certain chromosomal regions (44).These imprints provide the potential for monoallelicparent-of-origin-specific expression at certain times indevelopment or in specific cell types.

Normal imprinted gene expression is dependent ona normal relative number of specific parental alleles andrequires contributions from the alleles of both parents. Thatis, changing the number or proportion of parental alleles leadsto imprint deregulation. The most extreme example of changein the relative parental contributions occurs in conceptusesthat contain only maternal or only paternal genomic contri-butions. Only maternally derived chromosomes are found inmature cystic ovarian teratomas (MCT), which arise froma meiotic error during oocyte maturation (gynogeneticconceptus). The result is the formation of a cyst containingtissues from each of the three embryonic germ cell layers. Incontrast, an androgenetic conceptus, carrying two paternalgenomes and no maternal genome in all cells, fails to driveembryonic/fetal development and results in a hydatidiformmole (AnCHM). MCT and AnCHM display the seriousbiological consequences of uniparental inheritance (45), dem-onstrating that both parental genomes are required fornormal development of an embryo, with the paternal genomebeing required for extraembryonic tissues and the maternalgenome being required for embryonic development.

Most autosomal genes are normally expressed from bothpaternal and maternal alleles, whereas imprinted genes areexpressed predominantly, or exclusively, from either thematernal or paternal allele in a parent-of-origin-specificmanner. That is, an imprinted gene is expressed from thepaternal allele while the maternal copy is silenced, or,conversely, it is expressed from the maternal allele whilethe paternal allele is silenced (46). Imprinted genes do notconform to Mendelian genetics and do not predictparent-of-origin specificity for gene expression (47). About100 human genes have been reported to be imprinted

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FIGURE 2

DNA methylation reprogramming in preimplantation embryos. Soonafter fertilization both paternal (blue) and maternal (pink) pronuclei

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(http://igc.otago.ac.nz/home.html). Many such genes playvital roles in embryonic, fetal, and placental growth, as wellas in neurodevelopment (47–49).

Imprinted genes tend to cluster together to formimprinted domains. In humans, imprinted domains havebeen found on chromosomes 6, 7, 11, 14, 15, and 20(49–51). Within these domains, imprinted genes areregulated by imprinting centers (ICs). ICs are characterizedby the presence of differentially methylated regions (DMRs).Such DMRs carry parent-of-origin-specific DNA methylationand histone modification marks. These cis-acting DMRs,along with trans-acting factors, form the basis of theparent-of-origin-specific gene expression of imprinted genes.For example, the insulin growth factor2 (IGF2/H19) IC core-gulates the paternal expression of IGF2 with the maternalexpression of H19, two genes located in the same imprinteddomain 90 kb apart (52).

undergo genome-wide demethylation. Although demethylation ofthe maternal genome lags behind the paternal genome, both areremethylated around the time of implantation (yellow arrow). Incontrast to most other genes, epigenetic marks, such as DNAmethylation, at imprinted loci (ICs) are established early in germ linedevelopment and are protected from the global wave ofdemethylation early in embryonic development by maintenanceDNMT isoforms Dnmt1o, Dnmt1, and several other genes such asNLPR2, NLPR7, and Zfp57. The epigenetic reprogramming of thepreimplantation embryo also includes X inactivation. Notably, ARTtakes place during developmental time periods that involveepigenetic reprogramming. Adapted from reference (109).Inbar-Feigenberg. Basic concepts of epigenetics. Fertil Steril 2013.

Epigenetic Reprogramming in MammalianDevelopment

Our current knowledge about how the epigenome is reprog-rammed in early human development comes largely fromextensive research on mouse embryos. Genome-wide epigeneticreprogramming takes place at two pivotal developmental stages:during gametogenesis and during early embryogenesis. The firstevent occurs in the PGCs, where epigenetic marks, bothtissue-specificandparent-of-origin-specific imprints, are erased.New epigenetic marks, including parent-of-origin-specificimprints, are established at specific developmental time pointsboth before and after fertilization. After fertilization, a secondwave of reprogramming of epigenetic marks occurs in the earlyembryo, including global demethylation followed by de novoDNA methylation to allow for totipotency and subsequentcell-specific differentiation and lineage commitment (Fig. 2).Notably, epigenetic marks at ICs are protected from this waveof genome-wide epigenetic reprogramming.

Epigenetic Reprogramming in PGCs

Genome-wide erasure and reprogrammingof epigeneticmarksis initiated in the PGCs. The sex of the developing embryodictates which gamete, the egg or the sperm, will developfrom such early precursors. This sex-specific differentiationincludes the establishment of parent-of-origin-specificimprints in the sperm and oocytes.

Studies in mice have shown that PGCs begin theirmigration toward the developing genital ridge at day 8.5 ofembryonic life in mouse (E8.5), reaching their end point byE11.5 (53). Genome-wide demethylation of the PGCs isthought to occur mainly between E11.5 and E13.5, at thesite of the future gonads (54). The decrease in methylationis global; only 7% of CpGs remain methylated comparedwith 70%–80% in embryonic stem cells (ES) and somatic cells(25). Germ cell–specific gene promoters are methylated inearly PGCs, become demethylated, and are expressed duringreprogramming (55). At this time as well, PGCs of femaleembryos down-regulate Xist RNA expression from theinactive X-chromosome (Xi) (56, 57) so that two equivalent

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X-chromosomes may participate in meiosis for theproduction of female gametes.

De novomethylation in PGCs is established by recruitmentof Dnmt3a and Dnmt3l (58). The KRAB zinc finger proteinZFP57 also appears to play an important role specifically in oo-cyte imprint establishment (50, 59). Interestingly, the timing ofgamete development differs betweenmale and female embryos.Spermatogenesis occurs prenatally, and the associatedpaternal-specific DNA methylation programming is completeby birth (60). The epigenetic reprogramming of oocytes occursmuch later than in sperm. It begins at puberty and is nearlycomplete in each oocyte at the time of ovulation (60).

Developmental Epigenetic Reprogramming in theEarly Embryo

Before fertilization, the sperm and the egg are highly special-ized, differing in their gene expression as dictated by distinctpatterns 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, thepaternal pronuclear genome undergoes rapid demethylation(Fig. 2) (62–68). Demethylation in the maternal genomefollows, such that at the 4-cell stage of the embryo,the DNA methylation status of the two parental genomesare equalized (68). Epigenetic marks at imprinted genes are

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protected by specific proteins (Fig. 2) from epigenetic reprog-ramming throughout the preimplantation development of theembryo (54).

Lineage Commitment

Lineage commitment is well underway in the blastocyst, withthe 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, possiblysilencing the genes responsible for early developmental regu-lation (69, 70). By the time cells become fully committed totheir lineage, they will have attained a distinct epigeneticsignature reflecting their phenotype, developmental history,and environmental influences (61). Interestingly, placentalcells, derived from the trophectoderm, remain relativelyhypomethylated, compared with the differentiated deriva-tives of the ICM (71), in keeping with the role of theplacenta in endometrial invasion as well as its limited needfor extensive differentiation and longevity (61).

Epigenetic Mark Maintenance

Once established, methylation and other epigenetic marks inboth somatic and germ line cells must be maintained overthe course of future cell divisions. Several genes have beenfound to play a major role in this maintenance process inaddition to DNMT1 and DNMT1o; these include NLPR2,NLPR7, and ZFP57 (59, 72–75). As mentioned earlier, theZFP57 protein functions in establishing new imprints inoocytes; it is also needed for maintenance of methylationmore globally (59).

Epigenetic Deregulation and Human Disease

Epigenetic deregulation can result from disturbances in epige-netic control mechanisms or from alterations in the epigeneticmarks themselves, either in imprinted or nonimprinted genes.The development of multiple organ systems can be disrupted

TABLE 1

Genetic and epigenetic alterations in BWS.

Geneticalterations Sequence changes

Mutation of thematernalCDKN1Callele chromosome11p15.5

Mutations inNLRP2 at 19q13.42

Epigeneticalterations Meth

Loss of methylation at IC2on the maternal chromosome

Gain of mmate

Combinedalteration P

Inbar-Feigenberg. Basic concepts of epigenetics. Fertil Steril 2013.

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by deregulation of epigenetic control mechanisms. Mutationsin proteins that establish, erase, or read epigenetic marks cannegatively affect the development of multiple organ systems,as can be seen in a variety of human genetic syndromessuch as ATRX (OMIM#301040; alpha thalassemia, mental re-tardation) (12), Rubinstein-Taybi (OMIM#180849; CREBBP/EP300) (76), CHARGE (OMIM#214800; CHD7) (77), and Rettsyndromes (OMIM#312750; MECP2) (78).

An epigenetic alteration or ‘‘epimutation’’ refers to anaberrant DNA methylation or histone modification pattern.Such alterations occur in the absence or presence of anunderlying genomic alteration and are called primary andsecondary epimutations, respectively. A primary epimutationcan result from aberrant erasure, establishment, or mainte-nance of epigenetic marks (50).

In nonimprinted genes, somatic epimutations caused bymitotic errors in the normal maintenance of epigenetic markscan result in abnormal growth regulation. For example,approximately 20% of sporadic breast cancer displays hyper-methylation of the BRCA1 promoter, often in combinationwith an inherited or sporadic mutation on the second allele(79). Reduced growth potential can also result from anepimutation. For example, promoter methylation of WNT2(Wingless-type MMTV integration site family, member 2) inplacenta is associated with reduced birth weight percentilein the neonate (80), demonstrating that a single epigenetichit may impact human growth phenotypes.

Imprinting and Human Disease

Imprinted genes are subject to more complex epigeneticregulation than nonimprinted genes, providing more oppor-tunities to acquire epigenetic errors. The relative parentalcontributions of specific imprinted regions across the genomecan be disrupted by a number of different mechanisms,including genetic and/or epigenetic alterations (Table 1).

Primary epimutations (i.e., those without associatedgenomic alterations) can arise from stochastic errors inimprint reprogramming. For example, failure to erase imprintswill result in a gamete of one sex carrying the imprints of theother sex. When this gamete proceeds to fertilization, the

Cytogenetic abnormalities

Submicroscopic genomicalteration (duplication/deletion)within chromosome 11p15.5

Duplication, inversion, ortranslocation of 11p15.5

ylation abnormalities

ethylation at IC1 on thernal chromosome

Methylation alterations at multipleimprinted loci

arent of origin changes of gene copies

Paternal UPD of 11p15

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resulting zygote will carry uniparental imprints on bothparental chromosomes. Failure to establish appropriateimprints in the sperm or oocyte may lead to various disordersof growth and development, including subfertility (81).

Genomic alterations that contribute to imprinting disordersinclude deletions and duplications of imprinted regions. Also as-sociated with imprinting disorders is uniparental disomy (UPD),in which both homologues of a chromosomal region/segmentare inherited from only one parent (46, 82), that is, there aretwo copies of an imprinted region from one parent and nonefrom the other. UPD of nonimprinted genomic regions isgenerally not associated with disease, whereas UPD at specificgenomic locations containing imprinted regions can result inimprinting disorders. The extent of UPD may range froma small genomic segment to the entire chromosome (83). Theincidence of UPD of any chromosome is estimated to be about1:3,500 live births (84). Since deletion or duplication of animprinted genomic region changes the relative parentalcontributions, it represents another mechanism by whichimprinted genes can be regulated.

Many imprinted genes are expressed in the placenta andalso function in fetal growth regulation and braindevelopment (44). Epigenetic and associated geneticaberrations at imprinted genes result in human diseases thatoften reflect aberrant growth and/or aberrant neurodevelop-ment. As outlined below, it is very common for individualimprinting disorders to demonstrate etiologic heterogeneity.Further, different molecular etiologies may demonstrateepigenotype/genotype-phenotype correlations.

Neurologic and psychiatric disorders in which deregulationof imprinted genes are involved and parent-of-origin effects areobserved (85) include Prader-Willi syndrome (PWS) andAngelman syndrome (AS), two distinct neurodevelopmentalsyndromes that both map to the imprinted gene cluster onchromosome 15q11-q13 (86). These syndromes serve asan important example of how parental origin of thegenomic/epigenomic lesion defines which imprinting disorderoccurs.Apaternal deletionof chromosome15q11-13 ormaternalUPD15q11-13 leads toPWS,whereasamaternaldeletionof chro-mosome 15q11-13 or paternal UPD15q11-13 leads to AS (50).

One of the best known pediatric growth disorders causedby 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 complicatedby polyhydramnios, enlarged placenta, a long thickenedumbilical cord, increased risk of premature delivery (87),and placental mesenchymal dysplasia (88). The molecularetiology of BWS includes two different primary epimutations:hypomethylation of the proximal IC (IC2) on the maternalchromosome and hypermethylation at the distal IC (IC1) onthe maternal chromosome. Hypermethylation at IC2 canalso occur as a secondary epimutation when there is anunderlying deletion of the maternal IC2 region. In cases ofBWS with paternal UPD of chromosome 11p15.5, genomicimprints at both IC1 and IC2 are affected (51). Patients with

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BWS demonstrate important epigenotype-phenotypecorrelations. For example, the highest tumor risks occur inBWS cases with molecular abnormalities that includederegulation of IC1.

Russell-Silver syndrome (RSS) is characterized by poorpre- and postnatal growth, dysmorphic features, and variablepresence of limb/body asymmetry and developmental delay.To date, two different epigenetic defects have been associatedwith RSS (89). The first is maternal UPD of chromosome 7occurring in about 10% of RSS cases (90).

The second is hypomethylation at the IC1 on the paternalchromosome 11p15.5, which occurs in �45% of cases (89,91–93). The latter form is essentially opposite, both inphenotype and epigenotype, to cases of BWS with gain ofmethylation of the same region. Interestingly, some menwith oligospermia have been found to lack paternalmethylation at the IGF2/H19 IC1 on chromosome 11p15.5in their sperm (94). Children who inherit this chromosomefrom their fathers are at risk for developing RSS (89).

Imprinting disorders can include imprinting errors atmultiple imprinted domains. For example, in both BWS andRSS, some patients exhibit loss of methylation not only atthe IC on chromosome 11p15-IC1 for RSS and IC2 for BWSbut also at other imprinted loci. The molecular basis of thismultilocus loss of methylation (MLOM) is not known for thesetwo conditions. However, MLOM also occurs in the imprint-ing disorder transient neonatal diabetes mellitus (74), wherethe primary imprinting defect occurs at PLAGL1 DMR onchromosome 6q24; associated LOM at other maternal DMRshave been reported (74, 75). The cause of this MLOMcondition has been shown to be homozygous mutations inthe ZFP57 gene. This gene normally encodes an oocyte-derived maternal factor that participates in preimplantationmaintenance of imprints at multiple loci.

Mutations in NLRP genes can also be associated withimprinting errors at multiple genomic loci. NLRP proteins aremembers of the CATERPILLER protein family that is involvedin inflammation andapoptosis (50).Mutations ofNLRP7 in theoocyte are associated with aberrant imprints at multiple loci(72), causing formation of partial hydatidiform mole, whichis termed ‘‘biparental’’ due to its oocyte and sperm origins.

Epigenetic Deregulation in Fetal Development

Early embryonic development has been shown to be a criticalperiod of susceptibility to epigenetic deregulation. Indeed,studies of children born after maternal starvation have shownthat it is thefirst trimester of pregnancy that is a critical periodfor epigenetic changes (3, 5, 94). Interestingly, children bornafter the use of ART have also been identified to havechanges in epigenetic modifications, which are describedbelow (95, 96).

ART

ART accounts for 1%–2% of live births in developed countries(95). In general, such technologies are highly successful, withfew reports of adverse outcomes. An increased incidence ofepigenetic errors leading to imprinting disorders such as

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BWS, RSS (96), and AS (95) is reported in some studies ofchildren conceived by ART. However, not all studies havesupported such associations (97). The variability in theoutcomes of such studies is likely due to the large numbersof variable factors that could affect epigenetic reprogram-ming. It has been shown that subfertility itself could beassociated with epigenetic errors in the sperm of oligospermicmales (81). Data from mice shows that epigenetic errors canalso arise from ovulation induction or in vitro culture ofembryos (98). We expect that future investigations of new re-productive technologies will elucidate specific risk factors as-sociated with ART. In addition, we expect that only someindividuals who undertake ART will be genetically susceptibleto 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 transgenerationalenvironmental effects will be an important focus of futureresearch. A documented example of the importance ofenvironment on the evolution of epigenetically based diseasearises from the Dutch and Chinese famine studies (3, 5, 94).For example, male offspring who were exposed to the Dutchfamine of 1944–45 within the first 10 weeks after conceptiondemonstrated persistent methylation changes at the imprintedIGF2 gene six decades later (6, 100). The historical faminesprovide powerful ‘‘natural experiments’’ in humans thatresulted in the discovery of epigenetic marks that may bemodified by the prenatal environment (99). Prenatal exposureto the famine was associated with various adverse metabolicand mental phenotypes later in life, including a higher bodymass index (100–102), elevated plasma lipids (103), increasedrisk of schizophrenia (100–102, 104, 105), and possiblecardiovascular disease (106).

Exposure to nutritional factors, environmental pollut-ants, and medications during pregnancy may also alter fetalepigenetic marks. For example, choline intake during preg-nancy was found to increase placental promoter methylationof the cortisol regulation genes CRH and NR31C, leading toimproved stress response in children by lowering cortisollevels 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 andpostnatal development in rats, resulting in changes consistentwith the biology of primary ovarian insufficiency andpolycystic ovarian syndrome in humans (108). Some ofthese epigenetic changes were transmitted to the nextgeneration (109).

The role of epigenetic marks in translating the primarygenomic sequence has now moved to the forefront of humangenetic studies. Current epigenetic research is addressinga number of topics that will add to our understanding of therole of epigenetic mechanisms in human health and disease,including characterization of cis- and trans-acting influenceson the genetic background; delineating cell and tissue-specific epigenetic marks; determining the interactionsbetween epigenome and environment, with a focus on fetal

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programming; uncovering the effects of medication andnutrition; and assessing risks for adult-onset disorders.

REFERENCES1. Waterland RA, Michels KB. Epigenetic epidemiology of the developmental

origins hypothesis. Annu Rev Nutr 2007;27:363–88.2. Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, et al. Hotspots

of aberrant epigenomic reprogramming in human induced pluripotentstem cells. Nature 2011;471:68–73.

3. Huang C, Li Z, Wang M, Martorell R. Early life exposure to the 1959–1961Chinese famine has long-term health consequences. J Nutr;140:1874–8.

4. Mathers JC. Early nutrition: impact on epigenetics. Forum Nutr 2007;60:42–8.

5. Heijmans BT, Tobi EW, Stein AD, Putter H, Blauw GJ, Susser ES, et al.Persistent epigenetic differences associated with prenatal exposure tofamine in humans. Proc Natl Acad Sci U S A 2008;105:17046–9.

6. Chawla RK, Watson WH, Jones DP. Effect of hypoxia on hepatic DNAmethylation and tRNA methyltransferase in rat: similarities to effects ofmethyl-deficient diets. J Cell Biochem 1996;61:72–80.

7. Wellmann S, BettkoberM, Zelmer A, Seeger K, Faigle M, Eltzschig HK, et al.Hypoxia upregulates the histone demethylase JMJD1A via HIF-1. BiochemBiophys Res Commun 2008;372:892–7.

8. Yang J, Ledaki I, Turley H, Gatter KC, Montero JC, Li JL, et al. Role ofhypoxia-inducible factors in epigenetic regulation via histone demethy-lases. Ann N Y Acad Sci 2009;1177:185–97.

9. Otani J, Nankumo T, Arita K, Inamoto S, Ariyoshi M, Shirakawa M.Structural basis for recognition of H3K4 methylation status by the DNAmethyltransferase 3A ATRX-DNMT3-DNMT3L domain. EMBO Rep 2009;10:1235–41.

10. Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, et al.Distribution, silencing potential and evolutionary impact of promoterDNA methylation in the human genome. Nat Genet 2007;39:457–66.

11. The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 2004;306:636–40.

12. Maunakea AK, Chepelev I, Zhao K. Epigenome mapping in normal anddisease States. Circ Res 2010;107:327–39.

13. Lande-Diner L, Zhang J, Ben-Porath I, Amariglio N, Keshet I, Hecht M, et al.Role of DNA methylation in stable gene repression. J Biol Chem 2007;282:12194–200.

14. Ibrahim AE, Thorne NP, Baird K, Barbosa-Morais NL, Tavare S, Collins VP,et al. MMASS: an optimized array-based method for assessing CpG islandmethylation. Nucleic Acids Res 2006;34:e136.

15. Ziller MJ, Muller F, Liao J, Zhang Y, Gu H, Bock C, et al. Genomic distribu-tion and inter-sample variation of non-CpG methylation across human celltypes. PLoS Genet 2011;7:e1002389.

16. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev2002;16:6–21.

17. Costello JF, Plass C. Methylation matters. J Med Genet 2001;38:285–303.18. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and

Dnmt3b are essential for de novo methylation and mammalian develop-ment. Cell 1999;99:247–57.

19. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransfer-ase gene results in embryonic lethality. Cell 1992;69:915–26.

20. Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the estab-lishment of maternal genomic imprints. Science 2001;294:2536–9.

21. Pradhan S, Bacolla A, Wells RD, Roberts RJ. Recombinant humanDNA (cytosine-5) methyltransferase. I. Expression, purification, and com-parison of de novo and maintenance methylation. J Biol Chem 1999;274:33002–10.

22. Howell CY, Bestor TH, Ding F, Latham KE, Mertineit C, Trasler JM, et al.Genomic imprinting disrupted by a maternal effect mutation in theDnmt1 gene. Cell 2001;104:829–38.

23. Cedar H, Bergman Y. Programming of DNAmethylation patterns. Ann RevBiochem 2012;81:97–117.

24. Hajkova P, Jeffries SJ, Lee C, Miller N, Jackson SP, Surani MA. Genome-wide reprogramming in the mouse germ line entails the base excisionrepair pathway. Science 2010;329:78–82.

613

VIEWS AND REVIEWS

25. PoppC, DeanW, Feng S, Cokus SJ, Andrews S, Pellegrini M, et al. Genome-wide erasure of DNA methylation in mouse primordial germ cells isaffected by AID deficiency. Nature 2010;463:1101–5.

26. Gardner KE, Allis CD, Strahl BD. Operating on chromatin, a colorfullanguage where context matters. J Mol Biol 2011;409:36–46.

27. An integrated encyclopedia of DNA elements in the human genome.Nature 2012;489:57–74.

28. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A,et al. Landscape of transcription in human cells. Nature 2012;489:101–8.

29. KaikkonenMU, LamMT, Glass CK. Non-coding RNAs as regulators of geneexpression and epigenetics. Cardiovasc Res 2011;90:430–40.

30. Grewal SI. RNAi-dependent formation of heterochromatin and its diversefunctions. Curr Opin Genet Dev 2010;20:134–41.

31. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Droshainitiates microRNA processing. Nature 2003;425:415–9.

32. Hutvagner G, Zamore PD. AmicroRNA in a multiple-turnover RNAi enzymecomplex. Science 2002;297:2056–60.

33. Chuang JC, Jones PA. Epigenetics and microRNAs. Pediatr Res 2007;61:24R–9R.

34. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs andsiRNAs. Cell 2009;136:642–55.

35. Rosenbluth EM, Shelton DN, Sparks AET, Devor E, Christenson L, VanVoorhis BJ. MicroRNA expression in the human blastocyst. Fertil Steril2012; http://dx.doi.org/10.1016/j.fertnstert.2012.11.001.

36. Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A2009;106:11667–72.

37. GuttmanM, Amit I, GarberM, French C, LinMF, Feldser D, et al. Chromatinsignature reveals over a thousand highly conserved large non-coding RNAsin mammals. Nature 2009;458:223–7.

38. Sheik Mohamed J, Gaughwin PM, Lim B, Robson P, Lipovich L. Conservedlong noncoding RNAs transcriptionally regulated by Oct4 and Nanogmodulate pluripotency in mouse embryonic stem cells. Rna 2010;16:324–37.

39. Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncodingRNAs. Cell 2009;136:629–41.

40. Heard E, Disteche CM. Dosage compensation in mammals: fine-tuning theexpression of the X chromosome. Genes Dev 2006;20:1848–67.

41. Pessia E, Makino T, Bailly-Bechet M, McLysaght A, Marais GA. MammalianX chromosome inactivation evolved as a dosage-compensation mecha-nism for dosage-sensitive genes on the X chromosome. Proc Natl AcadSci U S A 2012;109:5346–51.

42. Morey C, Avner P. Genetics and epigenetics of the X chromosome. Ann NY Acad Sci 2010;1214:E18–33.

43. Okamoto I, Heard E. Lessons from comparative analysis of X-chromosomeinactivation in mammals. Chromosome Res 2009;17:659–69.

44. Reik W, Walter J. Genomic imprinting: parental influence on the genome.Nat Rev Gen 2001;2:21–32.

45. Mutter GL. Role of imprinting in abnormal human development. Mutat Res1997;396:141–7.

46. Choufani S, Shuman C,Weksberg R. Beckwith-Wiedemann syndrome. AmJ Med Genet C Semin Med Genet 2010;154C:343–54.

47. Tycko B, Morison IM. Physiological functions of imprinted genes. J CellPhysiol 2002;192:245–58.

48. Reik W, Constancia M, Fowden A, Anderson N, Dean W, Ferguson-Smith A, et al. Regulation of supply and demand for maternal nutrientsin mammals by imprinted genes. J Physiol 2003;547:35–44.

49. Weksberg R. Imprinted genes and human disease. Am J Med Genet CSemin Med Genet 2010;154C:317–20.

50. Horsthemke B. Mechanisms of imprint dysregulation. Am J Med Genet CSemin Med Genet 2010;154C:321–8.

51. Choufani S, Shapiro JS, Susiarjo M, Butcher DT, Grafodatskaya D,Lou Y, et al. A novel approach identifies new differentially methylatedregions (DMRs) associated with imprinted genes. Genome Res 2011;21:465–76.

614

52. Gabory A, Jammes H, Dandolo L. The H19 locus: role of an imprinted non-coding RNA in growth and development. Bioessays 2010;32:473–80.

53. McLaren A. Primordial germ cells in the mouse. Dev Biol 2003;262:1–15.54. Feng S, Jacobsen SE, Reik W. Epigenetic reprogramming in plant and ani-

mal development. Science 2010;330:622–7.55. Maatouk DM, Kellam LD, Mann MR, Lei H, Li E, Bartolomei MS, et al. DNA

methylation is a primary mechanism for silencing postmigratory primordialgerm cell genes in both germ cell and somatic cell lineages. Development2006;133:3411–8.

56. de Napoles M, Nesterova T, Brockdorff N. Early loss of Xist RNA expressionand inactive X chromosome associated chromatin modification in develop-ing primordial germ cells. PLoS ONE 2007;2:e860.

57. Sugimoto M, Abe K. X chromosome reactivation initiates in nascent pri-mordial germ cells in mice. PLoS Genet 2007;3:e116.

58. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, et al. Essential rolefor de novo DNA methyltransferase Dnmt3a in paternal and maternal im-printing. Nature 2004;429:900–3.

59. Li X, Ito M, Zhou F, Youngson N, Zuo X, Leder P, et al. A maternal-zygoticeffect gene, Zfp57, maintains both maternal and paternal imprints. DevCell 2008;15:547–57.

60. Saitou M, Kagiwada S, Kurimoto K. Epigenetic reprogramming in mousepre-implantation development and primordial germ cells. Development2012;139:15–31.

61. Morgan HD, Santos F, Green K, Dean W, Reik W. Epigenetic reprogram-ming in mammals. Hum Mol Genet 2005;14 Spec No 1:R47–58.

62. Santos F, Hendrich B, Reik W, Dean W. Dynamic reprogramming of DNAmethylation in the early mouse embryo. Dev Biol 2002;241:172–82.

63. Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of thezygotic paternal genome. Nature 2000;403:501–2.

64. Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, et al.Conservation of methylation reprogramming in mammalian development:aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A2001;98:13734–8.

65. Beaujean N, Hartshorne G, Cavilla J, Taylor J, Gardner J, Wilmut I, et al.Non-conservation of mammalian preimplantation methylation dynamics.Curr Biol 2004;14:R266–7.

66. Fulka H, Mrazek M, Tepla O, Fulka J Jr. DNA methylation pattern in humanzygotes and developing embryos. Reproduction 2004;128:703–8.

67. Lepikhov K, Wossidlo M, Arand J, Walter J. DNA methylation reprogram-ming and DNA repair in themouse zygote. Int J Dev Biol 2010;54:1565–74.

68. Hales BF, Grenier L, Lalancette C, Robaire B. Epigenetic programming: fromgametes to blastocyst. Birth Defects Res A Clin Mol Teratol 2011;91:652–65.

69. Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, et al. Dy-namic regulation of 5-hydroxymethylcytosine in mouse ES cells and duringdifferentiation. Nature 2011;473:398–402.

70. Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, et al. Tet1 andTet2 regulate 5-hydroxymethylcytosine production and cell lineage specifi-cation in mouse embryonic stem cells. Cell Stem Cell 2011;8:200–13.

71. Chapman V, Forrester L, Sanford J, Hastie N, Rossant J. Cell lineage-specificundermethylation of mouse repetitive DNA. Nature 1984;307:284–6.

72. Judson H, Hayward BE, Sheridan E, Bonthron DT. A global disorder of im-printing in the human female germ line. Nature 2002;416:539–42.

73. Meyer E, Lim D, Pasha S, Tee LJ, Rahman F, Yates JR, et al. Germline muta-tion in NLRP2 (NALP2) in a familial imprinting disorder (Beckwith-Wiede-mann Syndrome). PLoS Genet 2009;5:e1000423.

74. Mackay DJ, Callaway JL, Marks SM,White HE, Acerini CL, Boonen SE, et al.Hypomethylation of multiple imprinted loci in individuals with transientneonatal diabetes is associated with mutations in ZFP57. Nat Genet2008;40:949–51.

75. Mackay DJ, Temple IK. Transient neonatal diabetes mellitus type 1. Am JMed Genet C Semin Med Genet 2010;154C:335–42.

76. Munshi A, Shafi G, Aliya N, Jyothy A. Histone modifications dictate specificbiological readouts. J Genet Genom 2009;36:75–88.

77. SchnetzMP, Bartels CF, Shastri K, Balasubramanian D, Zentner GE, Balaji R,et al. Genomic distribution of CHD7 on chromatin tracks H3K4methylationpatterns. Genome research 2009;19:590–601.

VOL. 99 NO. 3 / MARCH 1, 2013

Fertility and Sterility®

78. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rettsyndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 1999;23:185–8.

79. Hansmann T, Pliushch G, Leubner M, Kroll P, Endt D, Gehrig A, et al.Constitutive promoter methylation of BRCA1 and RAD51C in patientswith familial ovarian cancer and early-onset sporadic breast cancer. HumMol Genet 2012;21:4669–79.

80. Ferreira JC, Choufani S, Grafodatskaya D, Butcher DT, Zhao C, Chitayat D,et al. WNT2 promoter methylation in human placenta is associated withlow birthweight percentile in the neonate. Epigenetics 2011;6:440–9.

81. Poplinski A, Tuttelmann F, Kanber D, Horsthemke B, Gromoll J. Idiopathicmale infertility is strongly associated with aberrant methylation of MESTand IGF2/H19 ICR1. Int J Androl;33:642–9.

82. Cassidy SB, Schwartz S, Miller JL, Driscoll DJ. Prader-Willi syndrome. GenetMed 2012;14:10–26.

83. Yamazawa K, Ogata T, Ferguson-Smith AC. Uniparental disomy andhuman disease: an overview. Am J Med Genet C Semin Med Genet2010;154C:329–34.

84. Robinson WP. Mechanisms leading to uniparental disomy and their clinicalconsequences. Bioessays 2000;22:452–9.

85. Isles AR, Wilkinson LS. Imprinted genes, cognition and behaviour. TrendsCogn Sci 2000;4:309–18.

86. Buiting K. Prader-Willi syndrome and Angelman syndrome. Am J MedGenet C Semin Med Genet 2010;154C:365–76.

87. Weng EY, Moeschler JB, Graham JM Jr. Longitudinal observations on 15children with Wiedemann-Beckwith syndrome. Am J Med Genet 1995;56:366–73.

88. Wilson M, Peters G, Bennetts B, McGillivray G, Wu ZH, Poon C, et al.The clinical phenotype of mosaicism for genome-wide paternal unipa-rental disomy: two new reports. Am J Med Genet A 2008;146A:137–48.

89. Eggermann T. Russell-Silver syndrome. Am J Med Genet C Semin MedGenet 2010;154C:355–64.

90. Monk D, Bentley L, Hitchins M, Myler RA, Clayton-Smith J, Ismail S,et al. Chromosome 7p disruptions in Silver Russell syndrome: delineat-ing an imprinted candidate gene region. Hum Genet 2002;111:376–87.

91. Horike S, Ferreira JC, Meguro-Horike M, Choufani S, Smith AC, Shuman C,et al. Screening of DNA methylation at the H19 promoter or the distalregion of its ICR1 ensures efficient detection of chromosome 11p15epimutations in Russell-Silver syndrome. Am J Med Genet A 2009;149A:2415–23.

92. Netchine I, Rossignol S, Dufourg MN, Azzi S, Rousseau A, Perin L, et al.11p15 imprinting center region 1 loss of methylation is a common andspecific cause of typical Russell-Silver syndrome: clinical scoring systemand epigenetic-phenotypic correlations. J Clin Endocrinol Metab 2007;92:3148–54.

93. Bartholdi D, Krajewska-Walasek M, Ounap K, Gaspar H, Chrzanowska KH,Ilyana H, et al. Epigenetic mutations of the imprinted IGF2-H19 domain in

VOL. 99 NO. 3 / MARCH 1, 2013

Silver-Russell syndrome (SRS): results from a large cohort of patients withSRS and SRS-like phenotypes. J Med Genet 2009;46:192–7.

94. Tobi EW, Slagboom PE, van Dongen J, Kremer D, Stein AD, Putter H, et al.Prenatal famine and genetic variation are independently and additivelyassociated with DNA methylation at regulatory loci within IGF2/H19.PLoS ONE 2012;7:e37933.

95. Hiura H, Okae H, Miyauchi N, Sato F, Sato A, Van De Pette M, et al.Characterization of DNA methylation errors in patients with imprintingdisorders conceived by assisted reproduction technologies. Hum Reprod2012;27:2541–8.

96. Eroglu A, Layman LC. Role of ART in imprinting disorders. Semin ReprodMed 2012;30:92–104.

97. Zheng HY, Shi XY, Wu FR, Wu YQ, Wang LL, Chen SL. Assisted reproduc-tive technologies do not increase risk of abnormal methylation of PEG1/M-EST in human early pregnancy loss. Fertil Steril;96:84–9.e2.

98. Denomme MM, Zhang L, Mann MR. Embryonic imprinting perturbationsdo not originate from superovulation-induced defects in DNA methylationacquisition. Fertil Steril;96:734–8.e2.

99. Heijmans BT, Tobi EW, Lumey LH, Slagboom PE. The epigenome: archive ofthe prenatal environment. Epigenetics 2009;4:526–31.

100. Li Y, He Y, Qi L, Jaddoe VW, Feskens EJ, Yang X, et al. Exposure to theChinese famine in early life and the risk of hyperglycemia and type 2 dia-betes in adulthood. Diabetes;59:2400–6.

101. Ravelli AC, van Der Meulen JH, Osmond C, Barker DJ, Bleker OP. Obesity atthe age of 50 y in men and women exposed to famine prenatally. Am J ClinNutr 1999;70:811–6.

102. Stein AD, Kahn HS, Rundle A, Zybert PA, van der Pal-de Bruin K, Lumey LH.Anthropometric measures in middle age after exposure to famine duringgestation: evidence from the Dutch famine. Am J Clin Nutr 2007;85:869–76.

103. Lumey LH, Stein AD. Transgenerational effects of prenatal exposure to theDutch famine. Br J Obstet Gynecol 2009;116:868. author reply.

104. Susser EB, Brown A, Matte TD. Prenatal factors and adult mental and phys-ical health. Can J Psychiatry 1999;44:326–34.

105. XuMQ, SunWS, Liu BX, Feng GY, Yu L, Yang L, et al. Prenatal malnutritionand adult schizophrenia: further evidence from the 1959-1961 Chinesefamine. Schizophr Bull 2009;35:568–76.

106. Painter RC, de Rooij SR, Bossuyt PM, Simmers TA, Osmond C, Barker DJ,et al. Early onset of coronary artery disease after prenatal exposure tothe Dutch famine. Am J Clin Nutr 2006;84:322–7, quiz 466–7.

107. Jiang X, Yan J, West AA, Perry CA, Malysheva OV, Devapatla S, et al.Maternal choline intake alters the epigenetic state of fetal cortisol-regulating genes in humans. Faseb J 2012;26:3563–74.

108. Nilsson EE, Anway MD, Stanfield J, Skinner MK. Transgenerationalepigenetic effects of the endocrine disruptor vinclozolin on preg-nancies and female adult onset disease. Reproduction 2008;135:713–21.

109. ReikW, DeanW,Walter J. Epigenetic reprogramming inmammalian devel-opment. Science 2001;293:1089–93.

615