This list is far from comprehensive and is meant to illustrate our point that significant phenotypic variation, including crossing a threshold to fatal disease, can emerge from animals that have an identical, cloned genetic background, and frequently-occurring differences in mitochondrial DNA cannot be a universal mechanism for a wide spectrum of phenotypic differences.
These early examples of cloned animals were subjected to intense scrutiny in highly supervised and controlled environments, yet they still exhibit disease in an inconsistent fashion. If environment were the source of this phenotypic variation, then one would expect the same emergence of disease among non-cloned members of this species, in an even greater extent, because their environment is not usually so tightly constrained.
More likely, there are other potential explanations for this variation. The general conclusion drawn from the previously described experiments is that substantial phenotypic variation may occur in the absence of either genetic background differences or identifiable environmental variation. When genetic sources of variation are excluded, environmental factors are usually considered to be the source of the remaining variation. However, the previously described data do not support this hypothesis.
It is easy to see how the environment is often blamed for this non-genetic variation in phenotype. It is difficult to prove that environmental factors are not affecting phenotype. Environmental sources of phenotypic variation can only be excluded by showing that variation persists in a zero-variation environment. Obviously it is difficult to design such an experiment in which environmental variation can be shown to be near-zero, but the studies described previously circumvent this problem.
They did so by either directly controlling the degree of environmental variation as in Gartner's experiments or by using naturally occurring human twins or artificially induced through in vitro embryo manipulations controls as comparison groups. In all of these examples, there exists a component of phenotypic variation whose source remains unexplained.
Epigenetics refers to DNA and chromatin modifications that play a critical role in regulation of various genomic functions. Although the genotype of most cells of a given organism is the same with the exception of gametes and the cells of the immune system , cellular phenotypes and functions differ radically, and this can be at least to some extent controlled by differential epigenetic regulation that is set up during cell differentiation and embryonic morphogenesis 26 — Even after the epigenomic profiles are established, a substantial degree of epigenetic variation can be generated during the mitotic divisions of a cell in the absence of any specific environmental factors.
Such variation is most likely to be the outcome of stochastic events in the somatic inheritance of epigenetic profiles. One example of stochastic epigenetic event is a failure of DNA methyltransferase to identify a post-replicative hemimethylated DNA sequence, which would result in loss of methylation signal in the next round of DNA replication reviewed in In tissue culture experiments, the fidelity of maintenance DNA methylation in mammalian cells was detected to be between 97 and Thus, the epigenetic status of genes and genomes varies quite dynamically when compared with the relatively static DNA sequence.
This partial epigenetic stability and the role of epigenetic regulation in orchestrating various genomic activities make epigenetics an attractive candidate molecular mechanism for phenotypic variation in genetically identical organisms. From the epigenetic point of view, phenotypic differences in MZ twins could result, in part, from their epigenetic differences. Because of the partial stability of epigenetic regulation, a substantial degree of epigenetic dissimilarity can be accumulated over millions of mitotic divisions of cells in genetically identical organisms.
The epigenetic defect is thought to arise from the unequal splitting of the inner cell mass containing the DNA methylation enzymes during twinning, which results in differential maintenance of imprinting at KCNQ1OT1.
In another twin study, the bisulfite DNA modification-based mapping of methylated cytosines revealed numerous subtle inter-individual epigenetic differences, which are likely to be a genome-wide phenomenon The finding that differences in MZT are similar to MZA, for a large number of traits, suggests that in such twins stochastic events may be a more important cause of phenotypic differences than specific environmental effects.
If the emphasis is shifted from environment to stochasticity, it may become clear why MZ twins reared apart are not more different from each other than MZ twins reared together.
It is possible that MZ twins are different for some traits, not because they are exposed to different environments but because those traits are determined by meta-stable epigenetic regulation on which environmental factors have only a modest impact. It is not our intention to argue that environment has no effect in generating phenotypic differences in genetically identical organisms.
Rather, we are suggesting that epigenetic studies of disease may help to understand the pathophysiology of, and susceptibility to, etiologically complex, common illnesses. The current method of studying most diseases includes molecular genetic approaches to identify gene-sequence variants that affect susceptibility and epidemiological efforts to identify environmental factors affecting either susceptibility or outcomes.
However, epidemiological studies in humans are limited by a number of methodological issues. Obviously, it is unethical to deliberately expose people to putative disease-causing agents in a prospective randomized controlled trial and it is impossible to control human environments in a way that eliminates most sources of bias in epidemiological studies Such designs may be possible in animal studies, but adequate animal models are available for only a small proportion of human conditions.
In this situation, epigenetic studies may help identification of the molecular effects of the environmental factors. There is an increasing list of environmental events that result in epigenetic changes 35 — 41 , including the recent finding of maternal behavior-induced epigenetic modification at the gene for the glucocorticoid receptor in animals The advantage of the epigenetic perspective is that, especially in humans, identification of molecular epigenetic effects of environmental factors might be easier and more efficient than direct but methodologically limited epidemiological studies.
Epigenetic mechanisms can easily be integrated into a model of phenotypic variation in multicellular organisms, which can explain some of the phenotypic differences among genetically identical organisms.
MZ twin discordance for complex, chronic, non-Mendelian disorders such as schizophrenia, multiple sclerosis or asthma could arise as a result of a chain of unfavorable epigenetic events in the affected twin. During embryogenesis, childhood and adolescence there is ample opportunity for multidirectional effects of tissue differentiation, stochastic factors, hormones and probably some external environmental factors nutrition, medications, addictions, etc.
Variation in phenotype among isogenic animals can also be attributed to meta-stable epigenetic regulation. Gartner's experiments with controlled versus chaotic environmental conditions showed that non-environmental factors were responsible for the majority of phenotypic variation among inbred animals. Similar observations among cloned animals can be accounted for by epigenetic differences among these animals. The role of dysregulated epigenetic mechanisms in disease is also consistent with the experimental observations in cloned animals.
The derivation of embryos from somatic cells, which contain quite different epigenetic profiles when compared with the germline, generates abnormalities of development that can arise from inadequate or inappropriate nuclear programming 22 , 44 , Evidence of epigenetic, non-environmentally mediated sources of variation in genetically identical organisms can be found in the examples of the mouse agouti and Axin Fu loci 46 , Transplantation experiments of fertilized oocytes to surrogate dams demonstrated that color was influenced by the phenotype of the genetic dam, not the foster dam Thus, an obvious phenotype of this isogenic mouse strain is controlled by epigenetic factors that are partially heritable.
Another example of the role of epigenetic mechanisms on phenotype is the murine axin-fused Axin Fu allele, which in some cases produces a characteristically kinked tail. Like the agouti gene locus, the Axin gene contains an intracisternal-A particle IAP retrotransposon that is subjected to epigenetic modification. The methylation status of the long terminal repeat of the IAP in the Axin Fu allele correlates with the degree of tail deformity. Furthermore, the presence of the deformity and associated methylation pattern in either sires or dams increases the probability of the same deformity in the offspring These experiments demonstrate both stochastic and heritable features of epigenetic mechanisms on variability in isogenic animals.
The two epigenetic mouse studies described previously as well as experimental data from other species 48 suggest epigenetic signals can exhibit meiotic stability, i. Traditionally, it has been thought that during the maturation of the germline, gametes re-program their epigenetic status by erasing the old and re-establishing a new epigenetic profile. Although the extent of meiotic epigenetic stability remains unknown, the implications are potentially dramatic, blurring the distinction between epigenetic and DNA sequence-based inheritance.
The inheritance of epigenetic information and the potential for this to affect disease susceptibility also challenge the dominant paradigm of human morbid genetics, which is almost exclusively concentrated on DNA sequence variation The variance within twin pairs s w 2 was significantly lower for isogenic MZ twins than for isogenic DZ twins because MZ twins derived from the same zygote shared the same epigenomic background.
DZ animals, however, originated from different zygotes that had different epigenetic backgrounds. This interpretation suggests that epigenetic meta-stability is not only limited to somatic cells but also applies to the germline and that germ cells of the same individuals may be carriers of different epigenomes despite their DNA sequence identity. Additionally, the inherited epigenetic signals have a significant impact on the phenotype despite numerous epigenetic changes that take place during embryogenesis 27 , Until recently, it has not been feasible to test these epigenetic interpretations of phenotypic differences directly among genetically identical organisms.
Technologies for high-throughput, large scale epigenomic profiling have been developed 51 — 55 , which along with more well-established techniques, such as focused fine-mapping of methylated cytosines using bisulfite modification or identification of histone modification 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 identical organisms to determine whether phenotypic differences are indeed correlated with differences in epigenetic profiles and where in the genome the crucial epigenetic signals may be located. The classical genetic phenomena of incomplete penetrance and variable expressivity may in part be explained by differences in epigenetic regulation of certain genes and their expression levels. We now have the experimental tools to test these hypotheses directly and characterize the extent to which epigenetic factors may influence the traditional dyad of genes and environment.
The source of phenotypic differences in genetically identical organisms may be one such blind spot among geneticists. Apart from human diseases, various concerns have been raised regarding the limitations of the DNA sequence-based paradigm, and the importance of epigenetic factors has been emphasized. In a similar way, Fedoroff et al.
As seen in other fields of science 59 , identification of the areas where inconsistencies or controversies lie may provide new opportunities for re-thinking fundamental laws, lead to new experimental designs, and may even result in major paradigmatic shifts.
We thank Dr Axel Schumacher for his help with drawing figures for this article. Figure 1. Figure 2. Epigenetic model of MZ twin discordance in complex disease, e. Red circles represent methylated cytosines. From the epigenetic point of view, phenotypic disease differences in MZ twins result from their epigenetic differences. Due to the partial stability of epigenetic signals, a substantial degree of epigenetic dissimilarity can be accumulated over millions of mitotic divisions of cells of MZ co-twins.
Although the figure shows that disease is caused by gene hypomethylation, scenarios where pathological condition is associated with gene hypermethylation are equally possible. Figure 3. Phenotypic differences in MZ isogenic animals A and DZ isogenic animals B can be explained by epigenetic variation in the germline. As in Figure 2 , red circles represent methyl groups attached to cytosines. Resnick, R. Macmillan, New York.
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Obstet Gynecol, , pp. MZ female twins discordant for X-linked diseases: a review. Acta Genet Med Gemellol Roma , 43 , pp. Wong, I. Phenotypic differences in genetically identical organisms: the epigenetic perspective. Hum Mol Genet, 14 , pp. Petronis, I. Gottesman, P. Kan, J. Kennedy, V. Basile, A. Paterson, et al. Monozygotic twins exhibit numerous epigenetic differences: clues to twin discordance?.
Schizophr Bull, 29 , pp. Bianchi, L. Integration of noninvasive DNA testing for aneuploidy into prenatal care: what has happened since the rubber met the road?. Clin Chem, 60 , pp. Kruyer, M. Mila, G. Glover, P. Carbonell, F. Ballesta, X. Am J Hum Genet, 54 , pp. Together, these findings suggest that methylation patterns can be to a large extent genetically determined and heritable, yet do not remain stable over a person's lifetime.
Variable degrees of epigenetic discordance can be seen in MZ twins, and it is evident that sample size, age, tissue type and CpG island selection can all significantly influence its estimates. There is substantial locus-to-locus and inter-individual variation in temporal methylation dynamics.
To date, there is conflicting evidence on early epigenetic discordance in MZ twins, but age should be a crucial factor in all future studies of methylome changes. Chorionicity seems to be an important factor altering discordance and heritability estimates, therefore studies investigating methylome concordance in twins should also take this into account, although this information is often lacking.
One potential problem that can affect findings in longitudinal studies is resampling from epigenetically different cellular subpopulations [ 87 ]. The environmental influence on the epigenome is relevant, and global epigenome studies in human MZ twins alone cannot resolve the sources of epigenetic discordance. Because the intrauterine environment, post-natal shared and non-shared environmental factors, and sequence polymorphisms acting in cis and trans can all be partly responsible for the methylation discordance, evaluating the significance of the intrinsic, stochastic epigenetic drift poses a methodological obstacle that is difficult to surmount [ 4 , 29 , 73 , 86 , 87 , 89 , 90 ].
Estimating the exact proportion of stochastically determined differences will require a deeper knowledge of the ways in which the non-shared environment shapes the methylome and the specific mechanisms responsible for the drift [ 29 ].
These may be difficult to investigate in humans. A number of studies have investigated methylation differences in MZ twins in relation to disease or different phenotypic conditions Table 2. Initially, studies utilized bisulfite conversion combined with sequencing of pre-selected candidate loci. In some of the very first twin methylation studies Petronis et al.
Another early study using bisulfite sequencing found methylation discordance at two regions of the COMT gene promoter in a sample of six MZ twin pairs, all discordant for birth weight [ 92 ]. Oates et al.
The proband had a significantly more methylated promoter than the healthy co-twin. The main limitations of these studies are the small sample sizes and narrow scope, limiting the number of potential associations to be found. With the advance of microarray and next-generation sequencing technology, it became possible to study methylation changes on a genome-wide scale. In a study of discordant risk-taking attitudes in a single MZ twin pair, Kaminsky et al looked at CpG methylation of about 12, CpG loci and found differences in methylation of the DLX1 gene, implicated in stress-response [ 94 ].
A methylation-sensitive-representational difference analysis study on a MZ pair discordant for bipolar disorder by Kuratomi et al. Using an Illumina GoldenGate array, Javierre et al looked at methylation of CpG sites in gene promoters across five MZ twins discordant for systemic lupus erythematosus SLE , five twins discordant for rheumatoid arthritis RA and five twins discordant for dermatomyositis DM , and discovered significant differences at 49 loci between the SLE-affected twins and their healthy co-twins, which were not seen in the RA and DM discordant twins [ 96 ].
The SLE cases had lower methylation levels and higher expression in several genes with immune functions. In a more recent analysis, Baranzini et al. The authors used high thresholds for methylation differences, which reduced the number of differentially methylated loci to 2, 10 and between the different twin pairs. The differences were inconsistent between the three twin pairs, leading the authors to conclude that methylation differences could not explain twin discordance. The study's small sample size and its heterogenous character twins of Ashkenazi Jewish African American and European descent constituted perhaps the greatest limitation reducing the power to detect significant methylation differences [ 97 ].
A recent analysis of methylation in three pairs of MZ twins discordant for autism using an 8. A slightly different approach was adopted by Mastreoni et al. Different methylation levels in temporal neocortex neuronal nuclei were found in two MZ twins discordant for Alzheimer disease, with hypomethylation in the affected twin [ 32 ]. Although genome-wide studies have enabled discovery of more DMRs, such studies are still in their infancy, and face a number of issues [ ]. To date, most studies have investigated methylation in small samples of one to a dozen twin pairs; use of larger discordant MZ twin cohorts will increase the power to detect potentially causal DMRs.
However, increasing the size of the twin sample might be challenging for rare diseases and study designs involving longitudinal sampling [ ]. Improvements to study designs in the future will probably require sampling from multiple tissues, particularly those that might be relevant to disease, because variation in the epigenome varies significantly across different cell types, and tissue-specific epimutations may play more important roles than systemic epimutations.
However, some tissues are not easily accessible, and sampling from different tissues might involve biopsy and post-mortem material [ ]. This is an important limitation, and some of the recent studies assayed methylation differences in tissues that were not directly relevant to the disease investigated.
Currently, the use of several technologies and platforms makes crosscomparisons difficult [ , ]. Comparisons between MZ and DZ and between MC and DC twins should provide insights into the role of genetics and intrauterine environment in shaping epigenetic variation. The associations yielded by various methylation studies emphasize the need to develop methods that establish causality [ , ].
Traditionally, in genetic studies, this was achieved by demonstrating perfect co-segregation of putative causal alleles with affected individuals in families, as well as alterations to the expression or structure of the protein encoded by the allele [ , ]. In non-mendelian complex diseases with a significant environmental component and no clear-cut disease segregation, causality is mainly investigated through case-control association studies by sorting candidate genes using P -value thresholds that minimize false-positive errors, and optimally by replicating the results in independent cohorts [ — ].
Of course, owing to linkage disequilibrium, population stratification, type I and type II errors, or mere chance, association does not equate to causality until proven by functional work [ , ]. Proving causality is, just like the definition, ultimately always context-dependent, and there is no uniform agreement on what constitutes adequate evidence; however, most authors are clear that some physical, biological link ought to be established [ , — ].
Epigenetic alterations at promoter sites should affect transcriptional activity [ 94 , 98 ]. However, the problem is that epigenetic differences could in fact be side-effects of disease or treatment. Studies investigating epigenetic changes are potentially prone to false conclusions as a result of reverse causation or confounding [ ].
Because the nature of the epigenome is dynamic and most epimutations arise throughout a person's lifetime, the key to addressing causality might be in their timing [ 97 ]. A longitudinal approach assaying for epigenetic discordance at birth or in early infancy and resampling at regular intervals should produce a timeline for methylation changes, and help to sort the potentially causal alterations from the secondary, side-effect ones.
Epigenetic differences identified in twins could be further investigated in longitudinal cohorts as part of a two-stage study design [ ]. Ultimately, studies of disease-associated epigenetic changes should be followed up with the aim of establishing a link with biological function [ ]. To date, only one longitudinal epigenome-wide study has been conducted, investigating single CpG methylation differences in a panel of MZ twins discordant for type 1 diabetes. The plausible assumption made by Galton[ ] that twin discordance can be explained by differential environmental exposures after birth is no longer tenable.
Genetics, the in utero environment, stochastity, and epigenetics can all potentially play a role in determining phenotypic discordance. The field of epigenetics is in its infancy. There is very strong evidence for the direct role and relevance of epigenetics in shaping human phenotypic variability.
The role of the epigenome can be both as a mediator of genetic and environmental effects or as an independent stochastic factor. Currently, the significance of primary epimutations in twin discordance is unknown. Furthermore, it is not fully clear as to what extent the epigenome is heritable and whether monozygotic twins are epigenetically identical at birth.
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