Phenotypic differences in genetically identicalorganisms: the epigenetic perspective

Phenotypic differences in genetically identicalorganisms: the epigenetic perspective

Albert H.C. Wong1, Irving I. Gottesman2 and Arturas Petronis1,*

1The Centre for Addiction and Mental Health, Departments of Psychiatry and Pharmacology, and the Institute of

Medical Science at the University of Toronto, Ontario, Canada and 2Department of Psychiatry and Psychology,

University of Minnesota, Minneapolis, MN, USA

Received January 26, 2005; Revised and Accepted February 24, 2005

Human monozygotic twins and other genetically identical organisms are almost always strikingly similar inappearance, yet they are often discordant for important phenotypes including complex diseases. Such vari-ation among organisms with virtually identical chromosomal DNA sequences has largely been attributed tothe effects of environment. Environmental factors can have a strong effect on some phenotypes, butevidence from both animal and human experiments suggests that the impact of environment has beenoverstated and that our views on the causes of phenotypic differences in genetically identical organismsrequire revision. New theoretical and experimental opportunities arise if epigenetic factors are consideredas part of the molecular control of phenotype. Epigenetic mechanisms may explain paradoxical findings intwin and inbred animal studies when phenotypic differences occur in the absence of observable envi-ronmental differences and also when environmental differences do not significantly increase the degree ofphenotypic variation.

INTRODUCTION

Identical human twins have been a source of superstition andfascination throughout human history, from Romulus andRemus, the mythical founders of Rome, to movies such asCronenberg’s ‘Dead Ringers’, to the twin paradox in thetheory of special relativity (1). For biologists and psycholo-gists, twins have been an important resource for exploringthe etiology of disease and for understanding the role ofgenetic and environmental factors in determining phenotype,and this fundamental question was first enunciated in itsalliterated form by Galton in the 19th century (2). The relativecontribution of nature versus nurture can be estimated by com-paring the degree of phenotypic similarities in monozygotic(MZ) versus dizygotic (DZ) twins. MZ twins arise from thesame zygote, whereas dizygotic twins arise from a pair ofseparate eggs, fertilized by two different sperm. As a result,MZ twins have the same chromosomal DNA sequence,except for very small errors of DNA replication after thefour to eight cell zygote stage. MZ twins share all of theirnuclear DNA, whereas DZ twins share only 50% of DNAsequence variation, on average. Therefore, the degree of

genetic contribution to a given phenotype can be estimatedfrom the comparison of MZ to DZ concordance rates orintra-class correlation coefficients. Traits that show higherMZ versus DZ similarity are assumed to have a genetic com-ponent because the degree of genetic sharing and the degree ofphenotypic similarity are correlated. The amount of geneticcontribution can be expressed as the ‘heritability’ (h2),which is calculated in various ways (e.g. as twice the differ-ence between the MZ and DZ concordance rates) (3,4).

Most (if not all) common human diseases show a significantheritability by this definition; however, while MZ twins appearvirtually identical, they are often discordant for disease. Forexample, the heritability of schizophrenia is variously reportedto be in the range of 0.70–0.84, on the basis of an MZ twinconcordance of �50% and a DZ twin concordance of10–15% (5–7). A heritability figure of 0.8 (or 80%) seemsto imply that the genetic contribution to disease susceptibilityis the main component of risk. Yet, half of MZ twin pairs, inthe case of schizophrenia, do not share the disease. The situ-ation is similar for virtually all complex non-Mendeliandiseases in which there is clearly some appreciable degreeof heritable risk, yet a significant proportion of MZ twin

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*To whom corresponding should be addressed at: The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, 250 CollegeStreet, Toronto, ON M5T 1R8, Ontario, Canada. Tel: þ1 4165358501; Fax: þ 1 4169794666; Email: [email protected]

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pairs is discordant for the disease. Figure 1 shows the MZ andDZ concordance rates for some common behavioral andmedical disorders.

Although heredity clearly influences disease risk, thesubstantial discordance between MZ twins indicates that chro-mosomal DNA sequence alone cannot completely determinesusceptibility (8). The imperfect disease concordance in MZtwins is an example of a more general phenomenon: (i.e. phe-notypic differences between or among genetically identicalorganisms). These differences have usually been attributedto the effects of environment (the ‘non-shared environment’in the case of MZ twins) (9), as a default explanation forvariation that remains after genetic effects are accountedfor. It is not easy to measure empirically the amount of non-genetic variation that is due to environmental factors. Thereare examples of significant environmental effects on diseaserisk, such as smoking and lung cancer (10), but directevidence of other measured environmental effects on pheno-type is rare. In addition, there is an increasing body of exper-imental evidence suggesting that the generally acceptedassumption—variation not attributable to genetic factors musttherefore be environmental—may require revision. Thisarticle will review human twin and animal data that highlightparadoxical findings regarding the contribution of heredityand environment to phenotype, followed by a reinterpretationof these experiments that incorporates epigenetic factors.

STUDIES OF MZ TWINS RAISED APART OR

TOGETHER: THE EPHEMERAL ROLE OF

ENVIRONMENT

One of the landmark studies in human twin research that chal-lenges the received importance of environment is the Minne-sota Study of Twins Reared Apart, in which detailedphysical and psychological assessments were conducted long-itudinally in over 100 MZ and DZ pairs of twins who had beenreared apart since early childhood (11,12). In a variation of thetraditional twin study comparing MZ twins reared together(MZT) with DZ twins reared together, the Minnesota studycompares MZT with MZ twins raised apart (MZA). Thisstudy design allows comparisons between genetically identicalMZ twin pairs who have been raised in a shared environment,at least as similar as for any two siblings, and those who havebeen raised in different homes, cities and states. Thus, thedegree of dissimilarity between the MZT and the MZA pairscan be assumed to be the result of different environments(13). A series of tests were administered simultaneously toeach pair of MZA and MZT twins, and the correlations oftheir scores on each scale were calculated and comparedwith test–retest correlations as a measure of the reliabilityof each scale. The intra-class correlation (R ) within pairs ofMZA (RMZA) and MZT (RMZT) was then expressed as aratio (RMZA/RMZT). Surprisingly, the correlations withinMZT and MZA twin pairs on personality measurementswere almost identical (e.g. RMZA ¼ 0.50 and RMZT ¼ 0.49on the Multidimensional Personality Questionnaire—MPQ).The RMZA/RMZT ratio for the MPQ was 1.02, compared with1.01 for fingerprint ridge counts. Out of 22 measurementsfor which the RMZA/RMZT ratio was reported, 15 had a value

over 0.9. In addition to the traits mentioned previously,these included: electroencephalographic patterns; systolicblood pressure; heart rate; electrodermal response (EDR)amplitude in males and number of EDR trials to reach habitu-ation; the performance scale on the WAIS-IQ; the RavenMill-Hill IQ test; the California Psychological Inventory;social attitudes on religious and non-religious scales andvarious scales of MPQ (11,12).

The findings of the Minnesota study are generally consistentwith other studies of MZA twins. For example, a recent effortto look at the etiology of migraine headaches gathered datafrom the Swedish Twin Registry and found that susceptibilityto migraine was mostly inherited and that the twins separatedearlier had even greater similarity in migraine status.For women in particular, migraine profiles were very similarin MZA compared to the MZT group, RMZA ¼ 0.58 andRMZT ¼ 0.46 (RMZA/RMZT ¼ 1.26), whereas for men, themigraine incidence was too low and the confidence intervalsfor correlations were too wide to be able to draw firm con-clusions (14). The Swedish Twin sample was also utilized toexplore the factors affecting regular tobacco smoking, andagain the results were similar for the two groups of MZ maletwins, RMZA ¼ 0.84 and RMZT ¼ 0.83 (RMZA/RMZT ¼ 1.01).For women, the data were more difficult to interpret,RMZA ¼ 0.44 and RMZT ¼ 0.68 (RMZA/RMZT ¼ 0.65), butwhen more recent cohorts were examined, the rates ofsmoking in women were similar to men and the heritabilitywas much the same as for men. Smoking rates in women inthe early 1900s (the oldest cohort in this sample) were verylow and social factors inhibiting smoking in women could

Figure 1. The MZ and DZ concordance rates for (A) some common beha-vioral (5–7,60–63) and (B) medical disorders (64–70). The concordancerates (%) shown are an approximate mid-range value derived from multiplereported figures.

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result in there being strong local environmental effects, whereasmore modern cohorts, with less restrictions on acceptablefemale behavior, were free to smoke for the same reasons(genetic) as men (15).

Peptic ulcer, a disease that contains an evident environ-mental component (i.e. exposure to Helicobacter pylori ),has also been subjected to an MZT/MZA study design. Theresults are another example of paradoxical findings in whichthe contribution of environmental and genetic factors isunclear. Again, using the Swedish Twin Registry, MZ andDZ concordance rates in twins raised together for pepticulcer were reported to be 0.65 and 0.35, respectively,suggesting that genetic factors are indeed important in vulne-rability (heritability 0.62). However, comparisons betweenMZA or MZT showed RMZA ¼ 0.67 and RMZT ¼ 0.65(RMZA/RMZT ¼ 1.03), suggesting that the common homeenvironment has little effect on susceptibility to peptic ulcer(16). So, the question that remains after analyzing these datais: what can account for the discordance in MZ twin pairs?If environment were a significant factor, then why is theRMZA/RMZT almost 1 (1.03)?

All the aforementioned examples of the MZT/MZA com-parisons, from anthropometric data to psychological traits topathogen susceptibility, lead to a paradoxical conclusionregarding the role of environmental factors. The fact that theMZ twin concordance (correlation) is well below 100%argues that environment is important. However, differentenvironments in MZA do not result in a higher degree ofphenotypic discordance when compared with MZT. TheseMZT/MZA studies clearly illustrate the core problem thatwe wish to explore in this paper; though some studies impli-cate non-genetic factors in susceptibility to disease or otherphenotypes, the available data do not support the interpretationthat the remaining variation in phenotype is due to environ-mental factors. Similar inconsistencies regarding the impactof environmental effects have also been detected in thestudies of experimental animals.

PHENOTYPIC VARIATION IN GENETICALLY

IDENTICAL ANIMALS: ‘THE THIRD COMPONENT’

Some of the questions raised by human twin studies can bere-examined by experimental manipulations of laboratoryanimals. Animal strains that have been inbred for many gene-rations have almost identical genomes, that is, they arevirtually isogenic. True MZ twins can also be generatedthrough in vitro embryo manipulations that provide an oppor-tunity to directly separate the effects of genes from pre-natalenvironment. Although environmental ‘twins’ do not exist inhumans, a close approximation can be created by strictlycontrolling the environment of laboratory animals in a waythat is impossible with humans. At the very least, the effectsof constrained versus diverse environments can be quantifiedto determine the relative contribution of specific environ-mental factors to phenotypic variation.

In an elegant series of experiments designed to explorethe relative contributions of genes, environment and otherfactors to laboratory animal phenotype, Gartner (17) wasable to demonstrate that the majority of random non-genetic

variability was not due to the environment. Genetic sourcesof variation were minimized by using inbred animals, butreduction of genetic variation did not substantially reducethe amount of observed variation in phenotypes such asbody weight or kidney size. Strict standardization of theenvironment within a laboratory did not have a majoreffect on inter-individual variability when compared withtremendous environmental variability in a natural setting.Only 20–30% of the variability could be attributed toenvironmental factors, with the remaining 70–80% of non-genetic variation due to a ‘third component . . . effective at orbefore fertilization’ (17).

To directly segregate genetic from pre- and post-nataleffects, in vitro embryo manipulations in isogenic animalscan be performed. In two mouse strains and in Friesiancattle, Gartner artificially created MZ and DZ twins by trans-planting divided and non-divided eight-cell embryos intopseudopregnant surrogates. The effect of different uterineenvironments was tested by transplanting pairs of MZ or DZembryos into the same or into two different surrogate dams.Pre-natal and post-natal environments were tightly controlledand, most importantly, were equally variable between theisogenic DZ and MZ twin pairs. The variance (s2) of meanbody weights and time to reach certain developmentalmilestones like eye opening, between twin pairs (sb

2) andwithin twin pairs (sw

2 ) was calculated for both MZ and DZgroups. The F-test comparisons for variation between theMZ and DZ groups overall were not significantly different.However, variance within twin pairs (sw

2 ) was signifi-cantly much greater for DZ twins than for MZ twins(ranging from P , 0.01 to P , 0.001 for individual weightmeasurements). Therefore, despite the fact that all micewere isogenic, and developed in identical pre- and post-natalenvironments, the MZ twin pairs showed a greater degreeof phenotypic similarity among co-twins than did the DZtwin pairs, thus implicating non-DNA sequence—and non-environment—based influences on the zygote at or beforethe eight-cell stage as the main source of phenotypic variation.Gartner and Baunack (18) referred to this non-genetic influ-ence as the ‘third component’, after genes and environment,the molecular basis of which remains unknown.

The cloning of mammals has recently been accomplished ina variety of species, and these experiments, technical feats inthemselves, also present an opportunity to differentiate theeffects of chromosomal DNA sequence from other factorsthat can influence phenotype. Although the offspring ofthese cloning experiments have the same genome as thedonor animals, they exhibit a variety of phenotypic abnormal-ities that obviously cannot be attributed to genetic causes (19).In some cases, the phenotypes are pathological and representdisease states, whereas other abnormalities are more subtle,suggesting that these observations are relevant to under-standing both susceptibility to human complex diseases andvariation within a normal functional spectrum (20). Themost famous of these cloning experiments was performedwith sheep, but along with seemingly healthy lambs, manyclone siblings died perinatally as a result of overgrowth,pulmonary hypertension and renal, hepatobiliary and body-wall defects (21). Some cloned mice are susceptible toobesity (22). In addition to higher overall weight, the cloned

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mice have the same inter-individual variability in weight asnon-cloned control mice of the same genetic background(23). Clones in other species also show considerable variationin lifespan and disease phenotypes between genetically identi-cal clones and non-cloned members of that species. This hasbeen reported in pigs (24) and in cattle, where the mainpost-natal abnormality is musculoskeletal in origin (25).

This list is far from comprehensive and is meant to illustrateour point that significant phenotypic variation, includingcrossing a threshold to fatal disease, can emerge fromanimals that have an identical, cloned genetic background,and frequently-occurring differences in mitochondrial DNAcannot be a universal mechanism for a wide spectrum of phe-notypic differences. These early examples of cloned animalswere subjected to intense scrutiny in highly supervised andcontrolled environments, yet they still exhibit disease in aninconsistent fashion. If environment were the source of thisphenotypic variation, then one would expect the same emer-gence of disease among non-cloned members of this species,in an even greater extent, because their environment is notusually so tightly constrained. More likely, there are otherpotential explanations for this variation.

The general conclusion drawn from the previouslydescribed experiments is that substantial phenotypic variationmay occur in the absence of either genetic background differ-ences or identifiable environmental variation. When geneticsources of variation are excluded, environmental factors areusually considered to be the source of the remaining variation.However, the previously described data do not support thishypothesis. It is easy to see how the environment is oftenblamed for this non-genetic variation in phenotype. It is diffi-cult to prove that environmental factors are not affecting phe-notype. Environmental sources of phenotypic variation canonly be excluded by showing that variation persists in azero-variation environment. Obviously it is difficult todesign such an experiment in which environmental variationcan be shown to be near-zero, but the studies described pre-viously circumvent this problem. They did so by eitherdirectly controlling the degree of environmental variation (asin Gartner’s experiments) or by using naturally occurring(human twins) or artificially induced (through in vitroembryo manipulations) controls as comparison groups. In allof these examples, there exists a component of phenotypicvariation whose source remains unexplained.

THE EPIGENETIC PERSPECTIVE

Epigenetics refers to DNA and chromatin modifications thatplay a critical role in regulation of various genomic functions.Although the genotype of most cells of a given organism is thesame (with the exception of gametes and the cells of theimmune system), cellular phenotypes and functions differradically, and this can be (at least to some extent) controlledby differential epigenetic regulation that is set up during celldifferentiation and embryonic morphogenesis (26–28). Oncethe cellular phenotype is established, genomes of somaticcells are ‘locked’ in tissue-specific patterns of gene expression,generation after generation. This heritability of epigeneticinformation in somatic cells has been called an ‘epigenetic

inheritance system’ (29). Even after the epigenomic profilesare established, a substantial degree of epigenetic variationcan be generated during the mitotic divisions of a cell in theabsence of any specific environmental factors. Such variationis most likely to be the outcome of stochastic events in thesomatic inheritance of epigenetic profiles. One example ofstochastic epigenetic event is a failure of DNA methyltransfer-ase to identify a post-replicative hemimethylated DNAsequence, which would result in loss of methylation signalin the next round of DNA replication (reviewed in 30). Intissue culture experiments, the fidelity of maintenance DNAmethylation in mammalian cells was detected to be between97 and 99.9% (31). In addition, there was also de novomethylation activity, which reached 3–5% per mitosis (31).Thus, the epigenetic status of genes and genomes variesquite dynamically when compared with the relatively staticDNA sequence. This partial epigenetic stability and the roleof epigenetic regulation in orchestrating various genomicactivities make epigenetics an attractive candidate molecularmechanism for phenotypic variation in genetically identicalorganisms.

From the epigenetic point of view, phenotypic differencesin MZ twins could result, in part, from their epigeneticdifferences. Because of the partial stability of epigenetic regu-lation, a substantial degree of epigenetic dissimilarity can beaccumulated over millions of mitotic divisions of cells ingenetically identical organisms. This is consistent withexperimental findings in MZ twins discordant for Beckwith–Wiedemann syndrome (BWS) (32). In skin fibroblasts fromfive MZ twin pairs discordant for BWS, the affected co-twins had an imprinting defect at the KCNQ1OT1 gene. Theepigenetic defect is thought to arise from the unequal splittingof the inner cell mass (containing the DNA methylationenzymes) during twinning, which results in differential main-tenance of imprinting at KCNQ1OT1. In another twin study,the bisulfite DNA modification-based mapping of methylatedcytosines revealed numerous subtle inter-individual epigeneticdifferences, which are likely to be a genome-wide phenom-enon (33). The finding that differences in MZT are similarto MZA, for a large number of traits, suggests that in suchtwins stochastic events may be a more important cause of phe-notypic differences than specific environmental effects. If theemphasis is shifted from environment to stochasticity, it maybecome clear why MZ twins reared apart are not more differ-ent from each other than MZ twins reared together. It is poss-ible that MZ twins are different for some traits, not becausethey are exposed to different environments but because thosetraits are determined by meta-stable epigenetic regulation onwhich environmental factors have only a modest impact.

It is not our intention to argue that environment has noeffect in generating phenotypic differences in genetically iden-tical organisms. Rather, we are suggesting that epigeneticstudies of disease may help to understand the pathophysiologyof, and susceptibility to, etiologically complex, common ill-nesses. The current method of studying most diseases includesmolecular genetic approaches to identify gene-sequence vari-ants that affect susceptibility and epidemiological efforts toidentify environmental factors affecting either susceptibilityor outcomes. However, epidemiological studies in humansare limited by a number of methodological issues. Obviously,




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it is unethical to deliberately expose people to putativedisease-causing agents in a prospective randomized controlledtrial and it is impossible to control human environments in away that eliminates most sources of bias in epidemiologicalstudies (34). Such designs may be possible in animal studies,but adequate animal models are available for only a smallproportion of human conditions. In this situation, epigeneticstudies may help identification of the molecular effectsof the environmental factors. There is an increasing list ofenvironmental events that result in epigenetic changes(35–41), including the recent finding of maternal behavior-induced epigenetic modification at the gene for the gluco-corticoid receptor in animals (42). The advantage of theepigenetic perspective is that, especially in humans, identifi-cation of molecular epigenetic effects of environmentalfactors might be easier and more efficient than direct (butmethodologically limited) epidemiological studies.

Epigenetic mechanisms can easily be integrated into amodel of phenotypic variation in multicellular organisms,which can explain some of the phenotypic differencesamong genetically identical organisms. MZ twin discordancefor complex, chronic, non-Mendelian disorders such asschizophrenia, multiple sclerosis or asthma could arise as aresult of a chain of unfavorable epigenetic events in theaffected twin. During embryogenesis, childhood and adoles-cence there is ample opportunity for multidirectional effectsof tissue differentiation, stochastic factors, hormones andprobably some external environmental factors (nutrition,medications, addictions, etc.) (39,43) to accumulate in onlyone of the two identical twins (Fig. 2) (30,33).

Variation in phenotype among isogenic animals can alsobe attributed to meta-stable epigenetic regulation. Gartner’sexperiments with controlled versus chaotic environmentalconditions showed that non-environmental factors wereresponsible for the majority of phenotypic variation amonginbred animals. Similar observations among cloned animalscan be accounted for by epigenetic differences among these

animals. The role of dysregulated epigenetic mechanisms indisease is also consistent with the experimental observationsin cloned animals. The derivation of embryos from somaticcells, which contain quite different epigenetic profiles whencompared with the germline, generates abnormalities of devel-opment that can arise from inadequate or inappropriate nuclearprogramming (22,44,45).

Evidence of epigenetic, non-environmentally mediatedsources of variation in genetically identical organisms canbe found in the examples of the mouse agouti and AxinFu

loci (46,47). The agouti gene (A ) is responsible for the coatcolor of wild-type mice and isogenic heterozygous c57BL/6Avy/a mice have a range of coat colors from yellow toblack (pseudoagouti). The darkness of the Avy/a mice wasproportional to the amount of DNA methylation in theagouti locus, with complete methylation in black psuedoagoutimice and reduced methylation in yellow ones (46). Transplan-tation experiments of fertilized oocytes to surrogate damsdemonstrated that color was influenced by the phenotypeof the genetic dam, not the foster dam (46). Thus, anobvious phenotype of this isogenic mouse strain is controlledby epigenetic factors that are partially heritable.

Another example of the role of epigenetic mechanismson phenotype is the murine axin-fused (AxinFu) allele,which in some cases produces a characteristically kinkedtail. Like the agouti gene locus, the Axin gene contains anintracisternal-A particle (IAP) retrotransposon that is sub-jected to epigenetic modification. The methylation statusof the long terminal repeat of the IAP in the AxinFu allelecorrelates with the degree of tail deformity. Furthermore, thepresence of the deformity and associated methylation patternin either sires or dams increases the probability of the samedeformity in the offspring (47). These experiments demon-strate both stochastic and heritable features of epigeneticmechanisms on variability in isogenic animals.

The two epigenetic mouse studies described previously aswell as experimental data from other species (48) suggest

Figure 2. Epigenetic model of MZ twin discordance in complex disease, e.g. schizophrenia. Red circles represent methylated cytosines. From the epigeneticpoint of view, phenotypic disease differences in MZ twins result from their epigenetic differences. Due to the partial stability of epigenetic signals, a substantialdegree of epigenetic dissimilarity can be accumulated over millions of mitotic divisions of cells of MZ co-twins. Although the figure shows that disease is causedby gene hypomethylation, scenarios where pathological condition is associated with gene hypermethylation are equally possible.




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epigenetic signals can exhibit meiotic stability, i.e. epigeneticinformation can be transmitted from one generation to another.Traditionally, it has been thought that during the maturation ofthe germline, gametes re-program their epigenetic status byerasing the old and re-establishing a new epigenetic profile.Although the extent of meiotic epigenetic stability remainsunknown, the implications are potentially dramatic, blurringthe distinction between epigenetic and DNA sequence-basedinheritance. The inheritance of epigenetic information andthe potential for this to affect disease susceptibility also chal-lenge the dominant paradigm of human morbid genetics,which is almost exclusively concentrated on DNA sequencevariation (49). This partial heritability of epigenetic status ofthe germline may explain the molecular origin of Gartner’s‘third component’ (Fig. 3). The variance within twin pairs(sw

2 ) was significantly lower for isogenic MZ twins than forisogenic DZ twins because MZ twins derived from the samezygote shared the same epigenomic background. DZanimals, however, originated from different zygotes that haddifferent epigenetic backgrounds. This interpretation suggeststhat epigenetic meta-stability is not only limited to somaticcells but also applies to the germline and that germ cells ofthe same individuals may be carriers of different epigenomesdespite their DNA sequence identity. Additionally, the inher-ited epigenetic signals have a significant impact on thephenotype despite numerous epigenetic changes that takeplace during embryogenesis (27,50).

Until recently, it has not been feasible to test these epige-netic interpretations of phenotypic differences directlyamong genetically identical organisms. Technologies forhigh-throughput, large scale epigenomic profiling have beendeveloped (51–55), which along with more well-establishedtechniques, such as focused fine-mapping of methylated cyto-sines using bisulfite modification or identification of histonemodification status using chromatin immunoprecipitation,can evaluate epigenetic profiles in a target tissue and permit

comparisons of epigenetic profile among different phenotypes.Methods such as these could be applied to genetically identicalorganisms to determine whether phenotypic differences areindeed correlated with differences in epigenetic profiles andwhere in the genome the crucial epigenetic signals may belocated. The classical genetic phenomena of incompletepenetrance and variable expressivity may in part be explainedby differences in epigenetic regulation of certain genes andtheir expression levels. We now have the experimentaltools to test these hypotheses directly and characterize theextent to which epigenetic factors may influence thetraditional dyad of genes and environment.

Vogel and Motulsky (56) wisely said that ‘human geneticsis by no way a completed and closed complex of theory . . .[with] results that only need to be supplemented in a straight-forward way and without major changes in conceptualiza-tion . . . anomalies and discrepancies may exist, but we oftendo not identify them because we share the ‘blind spots’ withother members of our paradigm group’ (56, p. 195). Thesource of phenotypic differences in genetically identicalorganisms may be one such blind spot among geneticists.Apart from human diseases, various concerns have beenraised regarding the limitations of the DNA sequence-basedparadigm, and the importance of epigenetic factors has beenemphasized. Strohman (57) concluded that the Watson–Crick genetic code ‘which began as a narrowly defined andproper theory and paradigm of the gene, has mistakenlyevolved into a theory and a paradigm of life’. In a similarway, Fedoroff et al. (58) stated that ‘our traditional geneticpicture . . .which is concerned almost exclusively with theeffect of nucleotide sequence changes on gene expressionand function is substantially incomplete’, and that ‘epigeneticfactors are significantly more important than it is generallythought’ (58). As seen in other fields of science (59), identifi-cation of the areas where inconsistencies or controversies liemay provide new opportunities for re-thinking fundamental

Figure 3. Epigenetic interpretation of Gartner’s ‘third component’. Phenotypic differences in MZ isogenic animals (A) and DZ isogenic animals (B) can beexplained by epigenetic variation in the germline. MZ animals derive from the same zygote and therefore their epigenetic ‘starting point’ is identical,whereas DZ animals originate from different sperm and oocytes that may carry quite different epigenomic profiles. As in Figure 2, red circles representmethyl groups attached to cytosines.




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laws, lead to new experimental designs, and may even result inmajor paradigmatic shifts.

ACKNOWLEDGEMENTS

We thank Dr Axel Schumacher for his help with drawingfigures for this article. This research has been supported bythe Special Initiative grant from the Ontario Mental HealthFoundation and also by NARSAD, the Canadian PsychiatricResearch Foundation, the Stanley Foundation, the JuvenileDiabetes Foundation International and the Crohn’s andColitis Foundation of Canada to A.P.

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Phenotypic differences in genetically identicalorganisms: the epigenetic perspective

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