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Epigenetic variation: amount, causes,and consequences (Genetics)

1. Introduction

The diversity of human phenotypes that we observe is the result of genetic andepigenetic variation and the interaction of these “biological” variables withenvironmental factors. Both large-scale and small-scale genome sequencing projects, aswell as more recent efforts to define structural variation (copy number variation andsubkaryotypic insertions, deletions and rearrangements), have resulted in an importantinitial description of the amount and type of genetic variation in the human genome. Onthe other hand, the scale of epigenetic variation in the human population is onlybeginning to be investigated.

Epigenetic variation may arise by diverse mechanisms but, at the molecular level, itreflects differences in the spatial configuration of chromatin and its interactions andfunction. Multiple biochemical processes (DNA methylation, histone methylation,acetylation, phosphorylation, sumoylation, etc.) are associated with these differences.One important consequence of this variability is the resultant variation in geneexpression, although many other effects have also been described (see the followingtext).

In the same way that somatic mutations can be transmitted through successive celldivisions, epigenetic marks can change during the lifespan of an organism and also betransmitted somatically through subsequent cell divisions. In fact, the normal phenotypicdiversity found between the different cell types of an organism is, with a few notableexceptions in the immune system, epigenetically controlled.

Interestingly, traits that result from particular patterns of epigenetic modification canalso be transmitted between generations in some circumstances. The term “epialleles”has been coined to describe such different epigenetic states (Article 36, Variableexpressivity and epigenetics, Volume 1). However, unlike DNA sequence changes,epigenetic modifications are often reversible at much higher frequencies than themutation rate. This is an important characteristic, because epigenetic marks can be resetbetween generations and they can change in response to the environment. Becauseepigenetic variation can also be genetically controlled, it constitutes a potentiallyimportant link between environmental and genetic factors (Cui etal., 1998; Nakagawaetal., 2001; Sandovici etal., 2003). Such a response to the environment could be mediatedby metabolic changes that result in epigenetic modifications (Paldi, 2003; Waterland andJirtle, 2004; Wolff et al., 1998). Consequently, epigenetic variability is not only a source ofphenotypic plasticity in response to the environment, but these epigenetic alterations canalso, potentially, be transmitted between generations, with very important implications in

Epigenetic variation: amount, causes, and consequences... http://what-when-how.com/genetics/epigenetic-variation...

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evolution (Rutherford and Henikoff, 2003; Sollars et al., 2003).

To better understand the relevance of epigenetic variation, we will discuss the extent(how much?), the origin (what are the causes?), and the implications (what are theconsequences?) of this important source of phenotypic variation.

2. Epigenetic variation: how much?

2.1. Epigenetic variation arises from multiple mechanisms

It is difficult to estimate the precise extent of epigenetic variation because itoccurs at multiple levels and as a result of multiple processes. The epigenetic variationresulting from inactivation of X chromosome provides a classic example of how multipleand distinct processes can give rise to very large fluctuation in phenotype amonggenetically similar or identical (Fraga et al., 2005) individuals. In human females (as inother female mammals), one of the two X chromosomes is inactivated by epigeneticmeans. Once one of the two X chromosomes is chosen for inactivation early indevelopment, and the same X chromosome remains inactive in all descendants of that cell(Article 41, Initiation of X-chromosome inacti-vation, Volume 1). The inactive Xchromosome becomes a cytologically visible heterochromatic body. This cytologicalmanifestation of femaleness (the Barr body) is to a large extent (but not completely(Disteche, 1995)) transcriptionally inert. This means that each single cell expresses onlyone allele of most (approximately 85%) (Carrel and Willard, 2005) X-linked genes. If bothX chromosomes have the same probability of being inactivated, the “average” women willhave the paternal X chromosome inactive in 50% of her cells and the maternal X inactivein the remaining 50% of her cells. However, because the process of choosing the Xchromosome for inactivation has a large stochastic component (Article 41, Initiation ofX-chromosome inactivation, Volume 1), individual women will have different patterns ofX-inactivation (Figure 1a). In fact, there is a minor fraction of females in whom >90% ofthe cells have the same X chromosome inactivated (Figure 1a). These females will havehighly preferential expression of either maternal or paternal alleles of all X-linked genesaffected by the inactivation process.

In addition to this partly stochastic, partly genetic variability in the fraction ofcells in which a particular X chromosome remains active (see next section), there is alsopopulation-level and intraindividual variability in the extent of X-inactivation. It has beendocumented that a fraction of X-linked genes have escaped inactivation’ (reviewed inDisteche, 1995). Interestingly, some genes are inactivated in some human samples, butescape inactivation in others (Carrel and Willard, 2005). In addition, the level ofexpression of such “escapees” also differs between samples (Carrel and Willard, 2005).Therefore, even genetically identical women (monozygotic twins) can differ in theirmosaic pattern of X-inactivation, the number of genes that escape X-inactivation, and thelevels of expression of some X-linked genes. In addition, some genes that have beeninactivated may become reactivated as a function of age (Wareham et al., 1987) or otherenvironmental factors, although not all X-linked genes appear equally susceptible toreactivation (Migeon et al., 1988; Pagani et al., 1990).


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Figure 1 Origin of X-inactivation variation. (a) Much of the variation in thehuman results from the stochastic component of the X-inactivation choiceprocess. Y-axis represents the fraction of women with the indicated percent ofcells with the same X chromosome active (Naumova et al., 1996 and ourunpublished data). Approximately one-third of women have either Xchromosomes inactivated in one-half of their of cells (purple bar) andapproximately 60% of women (purple bar plus green bar) have X-inactivationratios between 50:50 and 70:30. However, approximately 7% of women havehighly skewed patterns of X-inactivation, that is, greater than 90:10 (blue bar) infavor of the inactivation of a particular X chromosome. (b) Moving average ofchange in X-inactivation score in individual females (over nearly two decades;see Sandovici et al., 2004) as a function of age. Females who were greater than60 years of age when the first sample was taken show significantly morevariation over time than younger females. (c) Heritable effects on X-chromosomeinactivation variation. Distribution of X-inactivation ratios in heterozygous Xcea


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/Xcec mouse females – each circle represents an individual female mouse (de laCasa-Esperon et al., 2002). The X-controlling element locus affects theprobability that an X chromosome will become inactive, so that X chromosomescarrying the Xcea allele have a higher probability of being inactivated than Xchromosomes carrying the Xcec allele. The observed mean X-inactivation ratio ofthis population of females is 25% of cells with an active Xcea -carrying Xchromosome

A similar variability in a long-term inactivation phenomenon has been observedfor another class of monoallelically expressed genes located in the autosomes, theimprinted genes. Imprinted genes are expected to be expressed exclusively, or nearlyexclusively, from the paternal or the maternal copy (Article 37, Evolution of genomicimprinting in mammals, Volume 1). Several studies have shown that some imprintedgenes (e.g., IGF2, HTR2A genes) are expressed from both alleles in a small fraction ofnormal individuals (Bunzel etal., 1998; Sakatani et al., 2001), while others (IGF2R)exhibit the reciprocal characteristic of being imprinted in only a small fraction ofindividuals (Xu et al., 1993).

Expression levels between alleles have been found to be variable for severalimprinted genes in human tissues (Dao etal., 1998; McMinn etal., 2006), and have alsobeen observed at nonimprinted autosomal genes. In fact, large-scale transcriptionprofiling studies in humans have shown differential expression of alleles at a largeproportion of loci (up to 54%, depending on the cutoff level of differential expressionselected (Lo etal., 2003)) and, interestingly, the degree of difference in expressionbetween particular alleles varies between individuals (Lin etal., 2005; Lo etal., 2003; Pantetal., 2006; Pastinen etal., 2004). Moreover, skewing of allelic expression is notnecessarily in the same direction: in some individuals who are heterozygous for the samealleles, the allele that is preferentially expressed differs (Lo etal., 2003; Pastinen etal.,2004). This observation suggests that trans-modifiers and epigenetic variation areinvolved in the control of allelic differences in expression, in addition to polymorphisms incis-regulatory sequences. Such extensive variation in allelic expression must have a largeimpact in generating phenotypic diversity.

2.2. Variability in the biochemical “marks” associated with epigeneticvariation

The types of epigenetic marks that result in allelic variation in gene expression can beof diverse nature. The best known and most extensively investigated are covalentmodifications of DNA and core histones. DNA methylation at CpG sites shows a degree ofvariability between different individuals at multiple loci. This is the case for imprintedgenes like IGF2/H19 and IGF2R, for which interindividual variation in methylationpatterns has been observed in the differentially methylated regions associated with theirexpression (Sandovici etal., 2003). Interestingly, alterations in normal methylationpatterns of these regions have been associated with loss of imprinting (LOI), a commonobservation in several types of cancer (Cui et al., 1998; Nakagawa et al., 2001). Anotherinteresting example of interindividual variation at an imprinted gene is PEG1: this genecodes two isoform, one imprinted (isoform 1) and one expressed biallelically in multipletissues (isoform 2). However, in a large subset of human placentae, isoform 2 allelicexpression differences are observed, as well as interindividual variation in methylation ofan associated CpG island (McMinn et al., 2006).

Interindividual variability in methylation patterns has been also describedoutside of imprinted genes or even protein coding regions: this is the case of


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methylation differences between humans that is observed in specific Alu repeatedsequences (Sandovici et al., 2005). These observations reflect the fact that DNAmethylation may have roles in addition to transcriptional control (de la Casa-Esperon andSapienza, 2003; Pardo-Manuel de Villena etal., 2000; Sandovici etal., 2005).

Studies in other organisms also support the idea that variation in DNA methylationcould be a widespread phenomenon. For instance, variation in cytosine methylation hasbeen described in rRNA genes of natural accessions of the flowering plant Arabidopsisthaliana (Riddle and Richards, 2002), as well as in retrotransposons (Rangwala et al.,2006). Also, differentially methylated P1 pigment gene alleles have been observed inmaize (Das and Messing, 1994). Importantly, studies in Arabidopsis have also shown thatboth natural and induced methylation changes can be transmitted to the offspring andresult in developmental abnormalities in some instances (Kakutani et al., 1999; Rangwalaet al., 2006; Riddle and Richards, 2005).

3. Epigenetic variation: what are the causes?

Epigenetic variation is the result of three types of processes: stochastic,environmental, and heritable. Variation in X-inactivation illustrates all three of theseprocesses: during embryogenesis, one of the two X chromosomes is inactivated in eachcell and clonally transmitted through successive mitotic divisions. Because this choicehas a stochastic component (although some deterministic models are also capable ofexplaining the observations (Williams and Wu, 2004)), the X-inactivation patterns of apopulation of females approximates a normal distribution. The average female has abouthalf of her cells with the maternal X chromosome inactive and half with the paternal Xchromosome inactive. However, a small proportion of females show skewed patterns witha particular X chromosome being inactive in most cells (Figure 1a). Therefore, femalesare a mosaic for the expression of X-linked genes, and not even genetically identicalfemales need show the same mosaic pattern.

The so-called skewing of X-inactivation is not always the rare consequence of thestochastic nature of the choice process. In some instances, skewing is the result ofselection against X chromosomes carrying deleterious mutations, and the celltype-specificity of this skewing, as in X-linked agammaglobulinemia (skewing forinactivation of the mutant XLA/BTK allele in B-lymphocytes but not in T-lymphocytes),highlights the role of functional cellular selection (Fearon et al., 1987; reviewed inBelmont, 1996). In addition, skewing appears more common in older women, whichsuggests the contribution of environmental factors throughout their lifespan (Busque etal., 1996; Gale et al., 1997; Sharp et al., 2000). In this regard, X-inactivation seems toremain quite stable over many years during earlier ages (Sandovici et al., 2004) (Figure1b). Many older females, however, exhibit substantial changes over the timescales atwhich younger females do not exhibit changes (Sandovici et al., 2004). In this regard, wehave speculated (Sandovici et al., 2004) that acquired skewing of X-inactivation in olderfemales may result from discontinuous or catastrophic processes that result in decreasednumbers of stem cells or an age-related tendency toward bone marrow clonality ormyelodysplasia.

Additionally, preference for the inactivation of a particular X chromosome can have acompletely different origin compared to the selection for particular clonal cellpopulations or against disadvantageous mutations. Several studies in human and micehave shown that preference for X-inactivation can be heritable and genetically controlled


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(Cattanach and Isaacson, 1967; Naumova etal., 1996, 1998; Plenge et al., 1997) In themouse, the X-controlling element (Xce) is well known for its participation in theX-inactivation choice, so chromosomes carrying different alleles of Xce have differentprobabilities of being inactivated (Cattanach and Isaacson, 1967) (Figure 1c). Additionalautosomal loci also participate in the genetic control of the choice of the X chromosometo be inactivated in mice (Chadwick and Willard, 2005; Percec et al., 2002, 2003).Moreover, parent-of-origin effects have also been observed in both mice (Takagi andSasaki, 1975) and humans (Chadwick and Willard, 2005).

Stochastic, environmental, and genetic factors result in variability in X-chro-mosome inactivation and, consequently, generate a gamut of phenotypes for each ofthe X-linked genes, with multiple implications. The relative abundance of transcripts ofeach allele of any gene subject to X-inactivation reflects the fraction of cells with each ofthe two chromosomes active, as well as any allelic differences in expression that areintrinsic to specific alleles. Variations in such relative expression result in the spectrumof phenotypes observed in the population. For instance, a correlation betweenX-inactivation patterns and meiotic recombination levels (genomewide) has beendescribed in female mice (de la Casa-Esperon etal., 2002). The biological importance ofthis trait (recombination levels) in the human population cannot be overestimated as it isa major determinant of female fecundity and reproductive lifespan. If recombinationlevels are controlled by gene/s in the X chromosome, then levels of recombination canchange accordingly with the relative expression of different alleles of such gene/s.Because this is only one of the numerous genes in the X chromosome, the phenotypicdiversity generated by similar phenomena related to X-inactivation processes is expectedto be large in female mammals.

Similarly, epigenetic variability between individuals at multiple autosomal loci can bethe result of multiple processes. Since erasure and establishment of epigenetic marks is adynamic process that occurs during the lifespan of organisms, especially duringgametogenesis and embryogenesis (reviewed in Latham, 1999; Mann and Bartolomei,2002; Article 33, Epigenetic reprogramming in germ cells and preimplantation embryos,Volume 1), there is ample room for stochastic factors to contribute to the diversity ofpatterns observed. Environmental effects have also been described. Nutritional factorscan induce epigenetic modifications such as changes in the expression of imprintedgenes; moreover, maternal diet can affect the methylation status of transposableelements and the expression of nearby genes in mice (reviewed in Waterland and Jirtle,2004). Examples of environmental effects have also been reported in rats, in whichvariations in maternal care behavior result in epigenetic changes in the offspring at thelevel of histone acetylation and DNA methylation of the consensus sequence for theNGFI-A transcription factor of the glucocorticoid receptor gene. Consequently,expression of this gene in the hippocampus can be modified by maternal care, whichmight be the basis for the changes in stress response observed in this gene in theoffspring (reviewed in Fish etal., 2004). Environmental effects could be also the basis forthe changes observed in epigenetic marks over time. DNA methylation patterns changewith aging in a complex fashion, although overall hypomethylation has been observed inmost vertebrate tissues (Mays-Hoopes etal., 1986; Richardson, 2003). For instance,changes in the methylation profile of the c-myc proto-oncogene have been describedduring the aging process of mice. Because this is a gene involved in many tumorprocesses, similar temporal alterations of epigenetic marks might be part of the basis ofthe increasing incidence of cancer with age (Ono et al., 1986, 1989).

Finally, epigenetic diversity can be the result of heritable variants that affect theformation or stability of epigenetic marks. It has been observed that allelic differences in


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the expression of several genes are transmitted in families, although the patterns oftransmission are variable (Pastinen et al., 2004; Yan et al., 2002). In some instances, thetransmission of allelic imbalance is compatible with Mendelian inheritance, and evenassociated with transmission of particular polymorphisms (haplotypes), suggesting theparticipation of cis-acting elements in the regulation of allelic expression (Yan et al.,2002), whether they are of genetic or epigenetic origin. In fact, studies showingtransmission of de novo induced methylation changes indicate that chromatinmodifications, per se, are heritable (Kakutani et al., 1999; Stokes et al., 2002). Moreover,abnormal methylation patterns at the differentially methylated regions of the IGF2/H19and IGF2R imprinted genes have been found to cluster in families (Sandovici et al.,2003). Also, methylation levels at particular Alu repeated sequences show interindividualdifferences when the insertions were paternally versus maternally transmitted (Sandoviciet al., 2005). In the case of imprinting defects, epimutations in an imprinting controlregion of human chromosome 15 have been associated with a substantial percentage ofcases of the neurodevelopmental disorders Angelman and Prader-Willi syndromes (seeArticle 29, Imprinting in Prader-Willi and Angelman syndromes, Volume 1). Recentstudies have shown that both cis- and trans-acting factors seem to increase the risk ofconceiving a child with Angelman syndrome (AS) (Zogel et al., 2006). Trans-actinggenetic elements have also been involved in changes in the imprinting status of the Dlk1gene in mouse brain (Croteau et al., 2005). In this case, reactivation of the normallysilent maternal allele correlates with the methylation status of a differentially methylatedregion. Therefore, epigenetic information constitutes a code superimposed on the geneticinformation, thereby increasing phenotypic diversity. Much future research will no doubtfocus on determining whether epigenetic variation makes a significant contribution tocommon “complex genetic disorders”, such as diabetes, hypertension, schizophrenia,Alzheimer’s disease and the like, in humans.

4. Epigenetic variation: what are the consequences?

Phenotypic diversity is the direct consequence of much epigenetic variation. Aswe mentioned before, epigenetic modifications can result in allelic expression imbalancewithin (differential expression levels) or between cells (monoallelic and mosaicexpression). This, in turn, can result in phenotypic differences between cells, tissues,and/or individuals. The most obvious example is that of monozygotic twins: althoughgenetically identical, numerous phenotypic differences appear during their life span. Thesame is true at the epigenetic level: recent studies have shown that differences in DNAmethylation and histone acetylation between twins are present throughout the genome(Fraga et al., 2005). Therefore, epigenetic differences could be the basis of manyphenotypic discordances observed between twins, including their susceptibility tocomplex diseases (Wong et al., 2005).

4.1. Epigenetic variation and disease

Epigenetic variation is particularly important for genes involved in diseases. Forinstance, the fragile-X syndrome of mental retardation is associated with an expansion inthe number of CGG repeats in the promoter and 5′ untranslated region of the FMR1 geneon chromosome X. This expansion results in hypermethylation of the region and silencingof the FMR1 gene (Hansen et al., 1992). Short expansions (premutations) do not haveapparent phenotypic effects, while long expansions are observed in affected individuals.Notably, the severity of the disease ranges from severe mental retardation to only mildlearning disabilities. It is possible that the observed gamut of symptoms depends, at leastin part, on epigenetic differences, because variability in methylation in this region has


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been observed between and within individuals (Genc etal., 2000; Stoger etal., 1997) andchanges in the CGG repeat length might also result in additional chromatin andtranscriptional modifications.

Another interesting example of mosaicism has been observed in a small group ofAS patients, in whom an imprinting defect silences the maternal copy of the UBE3Agene. However, some of these patients show mosaic maternal expression and methylationof this gene, which, again, suggests the possibility of an epigenetic effect on the observedvariability in the severity of clinical symptoms (Nazlican et al., 2004).

Cancer has also been associated with epigenetic alterations, such as losses andgains of methylation and LOI (Feinberg et al., 1988, 2002; Cui et al., 2003; Jones andBaylin, 2002; Nakagawa et al., 2001). Interestingly, some of these alterations are alsoobserved in normal tissues of the same individuals, as highlighted by the gain of DNAmethylation in the imprinting control region upstream of H19 in human Wilms tumorsand in the non-neoplastic kidney parenchyma adjacent to these tumors (Cui et al., 1998,2003; Moulton et al., 1994). Hence, epigenetic variation between individuals is probablyinvolved in susceptibility to develop cancer as well as other genetic diseases. Moreover,since heritable epigenetic variation has been observed in many instances, it can actuallyplay an important role in quantitative trait variation, and selection acting on suchepialleles might result in rapid phenotypic changes, making it a formidable force inevolution (Rutherford and Henikoff, 2003; Sollars et al., 2003).

4.2. Epigenetic variation and development

Epigenetic variation also has important consequences in development anddifferentiation. A potentially important example of epigenetic changes as a result ofenvironmental effects is the effects of culture conditions on the expression of imprintedgenes in mouse embryos. It has been shown that some culture media perturbs geneexpression and results in aberrant methylation and expression of imprinted genes(Doherty et al., 2000; Mann et al., 2004; Rinaudo and Schultz, 2004). Although some ofthese abnormalities can be restored in the embryo proper (Mann et al., 2004), manypersist in the extraembryonic tissues and can potentially affect the development of theembryo. In fact, several epidemiological studies suggest that assisted reproductivetechnologies (ART) might result in an increased frequency of diseases caused byimprinting defects, such as AS and Beckwith-Wiedemann syndrome (BWS) (Article 30,Beckwith-Wiedemann syndrome, Volume 1).

Despite the many reassuring reports on the safety of ART, there have been a smallnumber of recent reports suggesting that ART children may be at increased risk for rarecongenital malformation syndromes that are related to defects in genome imprinting (Coxet al., 2002; DeBaun et al., 2003; Halliday et al., 2004; Horsthemke et al., 2003; Niemitzet al., 2004; Olivennes et al., 2001; Orstavik et al., 2003). At least three childrenconceived by intracytoplasmic sperm injection (ICSI) have been diagnosed with AS(Horsthemke et al., 2003; Orstavik et al., 2003) and at least 28 ART children (both invitro fertilization (IVF) and ICSI cases) have been diagnosed with BWS (Boerrigter et al.,2002; Bonduelle et al., 2002; DeBaun et al., 2003; Gicquel et al., 2003; Halliday et al.,2004; Koudstaal etal., 2000; Maher et al., 2003; Olivennes et al., 2001; Sutcliffe et al.,1995). Because both AS and BWS are rare disorders (each affects approximately 1 in 15000 children (Nicholls et al., 1998)), the appearance of even small numbers of cases isunexpected except among a large sample of births. Therefore, the current data stronglysuggests that there is an association between increased risk for AS and BWS and ART.With respect to BWS, the number of affected individuals observed is estimated to be up


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to nine times the expected incidence (Halliday et al., 2004).

The epidemiological assessment that ART may lead to an increase in the frequency ofdefective genome imprints is also supported by biochemical characterization of alleles atthe relevant disease loci. All three cases of AS show allelic DNA methylation patternscharacteristic of a sporadic imprinting defect at the AS locus (i.e., complete or mosaicabsence of methylation on both maternal and paternal alleles (Horsthemke et al., 2003;Orstavik et al., 2003)). None of the patients has a cytogenetically visible alteration ofchromosome 15 (which occurs in 70% of all AS cases (Nicholls et al., 1998)) and none hasa detectable microdeletion at the imprinting center, suggesting that all three cases aredue to sporadic, primary, epigenetic defects rather than genetic changes. Given that suchimprinting defects account for less than 5% of all AS cases (Buiting etal., 2001, 2003;Nicholls et al., 1998), there is at least a suspicion that all three cases occurring inpatients following ICSI are of this type.

The case for the presence of primary epigenetic defects in the majority of theBWS patients found among ART children is also supported by molecular analyses ofalleles at the BWS locus on chromosome 11. Nineteen of the 24 patients have beenanalyzed for “loss of imprinting” (“LOI”; defined, in this context, as transcription of bothmaternal and paternal alleles; or the specific changes in DNA methylation that track withthis phenomenon and provide a more robust marker in clinical samples) at one or moreimprinted genes within the BWS locus and 13 of the 19 cases showed LOI at eitherKCNQ10T1 (DeBaun et al., 2003; Gicquel etal., 2003; Maher etal., 2003) or H19/IGF2(DeBaun etal., 2003). With the addition of the BWS patients described by Halliday et al.,2004, 16 out of a total of 22 cases examined showed LOI. Although imprinting defects aremore common in BWS than in AS, LOI still appears to be overrepresented among BWScases in ART children and ART is, in turn, overrepresented among BWS cases.

4.3. Epigenetic variation diversity

During the last several years, there has been a dramatic increase in the number ofstudies attempting to elucidate the patterns and interrelationship between DNAmethylation, histone modifications, noncoding RNAs, binding of nonhistone chromatinproteins, nuclear positioning and interactions, and so on, which are part of the“epigenetic code” (Article 27, The histone code and epigenetic inheritance, Volume 1).Alterations of the chromatin configuration can affect interactions between DNA regions,between chromosomes, and with other molecules. Most of the studies in epigeneticvariation have been focused on the different mechanisms and effects on gene expressionand its phenotypic consequences, including allelic differences and disease, enhancersand insulators, trans-sensing and paramutation, long-range interactions and nuclearcolocation, and so on. However, epigenetic changes have also been found to affect manyother chromosomal functions (see the following text). A classical example is thecentromere, in which multiple chromatin modifications and proteins play a major role inbinding to the poles of the spindle and promoting chromosome segregation. Interestingly,epigenetic changes can generate new domains with similar properties (neocentromeres)that affect the segregation of chromosomes during mitosis and meiosis (Pardo-Manuel deVillena and Sapienza, 2001; Rhoades and Dempsey, 1966; Warburton, 2004).Consequently, changes in the segregation of chromosomes or chromatids can favor thetransmission of particular alleles to the next generations, with important consequences inevolution and disease (Pardo-Manuel de Villena and Sapienza, 2001).

Another example of a biochemical process for which there is a strong epigeneticeffect is asynchronous DNA replication. Asynchronous replication is characteristic of


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regions containing monoallelically expressed genes (Mostoslavsky et al., 2001; Simon etal., 1999) and, therefore, epigenetic differences seem to be the basis for the differentialreplication between homologs at such regions. Consequently, these chromosomal regionsare interesting examples of how epigenetic modifications of chromosomal regions havenot one but multiple effects (on replication and expression). In addition, a recent surveyof asynchronously replicated regions have found that they are located in close proximityto areas of tandem gene duplication (Gimelbrant and Chess, 2006) – although whethersuch epigenetic marks play a role in chromosome stability in regions of duplicationsremains to be determined.

Meiotic pairing and recombination constitute another example of a cellularprocess in which epigenetic marking appears to play an important role. Functional andepigenetic differences between paternal and maternal chromosomes are a commonobservation in sexually reproducing organisms (reviewed in de la Casa-Esperon andSapienza, 2003; Pardo-Manuel de Villena et al., 2000). However, only a few of suchdifferences have been associated with imprinted gene expression. Consequently, it hasbeen postulated that parent-of-origin epigenetic differences share a common origin andfunction in all sexually reproducing organisms: to allow the recognition (and distinction)between homologous chromosomes during the processes of recombination and repair (dela Casa-Esperon and Sapienza, 2003; Pardo-Manuel de Villena et al., 2000). Indeed, arecent study has shown that DNA methylation has a role in early meiotic stages: micedeficient in the DNA methyltransferase 3-like (Dnmt3L) gene are sterile and displayabnormal chromosome synapsis during meiosis (Bourc’his and Bestor, 2004). Curiously,normal expression of Dnmt3L occurs not in the meiotic cells, but in their precursors.Hence, the epigenetic signals must be inherited through multiple cell divisions. Suchepigenetic signals are observed as DNA methylation of retrotransposons, which appeardemethylated in Dnmt3L knockout male germ cells. While methylation participates in thenormal silencing of mobile elements, retrotransposons are transcribed in the mutantmice. Therefore, Dnmt3L mutant mice represent an example of how epigenetic changescan not only affect transcription but can also reshape the genome by affecting synapsisand allowing the mobilization of retrotransposons into new locations, with multipleconsequences. Consequently, studies of epigenetic variation cannot be restricted toeffects on gene expression, because it can also modulate many other chromosomefunctions (de la Casa-Esperon and Sapienza, 2003; Pardo-Manuel de Villena etal., 2000;Sandovici et al., 2005).

5. Conclusions

When discussing epigenetic variation, it is important to remember that we know littleabout either the underlying mechanisms or the consequences. To mention a few recentexamples, studies on the viable yellow allele of the mouse agouti locus (Avy) have shownthat the expression of the agouti gene is correlated with the methylation status ofupstream sequences (Article 36, Variable expressivity and epigenetics, Volume 1).Interestingly, epigenetic inheritance at this locus is not due to such methylation marks,because they are erased during embryonic development (Blewitt etal., 2006). Therefore,other epigenetic marks are responsible for the transmission of this epiallele to theoffspring. In this review, although we have mostly mentioned examples of variability inmethylation (because it has been the most frequently studied epigenetic mark inmammals and is the first subject of the Human Epigenome Project (Eckhardt et al.,2004)), we hope that current and future studies will bring to light epigenetic variation atmany other levels. For instance, studies of the effects of histone tail modifications atmultiple amino acid residues are an expanding field, because the spectrum of


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modifications and residues affected continues to grow (Article 27, The histone code andepigenetic inheritance, Volume 1). In addition, new epigenetic marks and inheritancemodes are likely to be discovered. For instance, the role of small RNAs on epigeneticchanges has become prominent since the discovery of RNA interference inCaenorhabditis elegans (Fire etal., 1998). Recent studies have revealed striking newroles for RNA in non-Mendelian epigenetic inheritance, similar to paramutation in plants.

The homozygous wild-type progeny of mice that are heterozygous for a mutation inthe Kit gene (Rassoulzadegan et al., 2006) are found to exhibit the white spottingphenotype that is characteristic of mice that carry a Kit mutation. Elaboration of thisphenotype is related to the zygotic inheritance of abnormally processed RNAs of thenormal allele.

A realistic description of the scale of epigenetic variation is hampered by thediversity of causes and consequences and because the mechanism by which manyepigenetic marks are heritable remains obscure. An increasing number of studies areaiming to integrate profiles from different epigenetic marks and gene expression patternsof particular chromosomal regions, in order to better understand the possibilities ofvariations on the epigenetic code. The complexity and diversity of the epigenetic marksand their implications poses a tremendous challenge, but understanding the nature of theimmense phenotypic diversity that surrounds us makes it worth the effort.

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