A twin approach to unraveling epigenetics
Authors: Jordana T. Bell, Tim D. Spector
Journal: Trends in Genetics (2011)

The regulation of gene expression plays a pivotal role in complex phenotypes, and epigenetic mechanisms such as DNA methylation are essential to this process. The availability of next-generation sequencing technologies allows us to study epigenetic variation at an unprecedented level of resolution. Even so, our understanding of the underlying sources of epigenetic variability remains limited. Twin studies have played an essential role in estimating phenotypic heritability, and these now offer an opportunity to study epigenetic variation as a dynamic quantitative trait. High monozygotic twin discordance rates for common diseases suggest that unexplained environmental or epigenetic factors could be involved. Recent genome-wide epigenetic studies in disease-discordant monozygotic twins emphasize the power of this design to successfully identify epigenetic changes associated with complex traits. We describe how large-scale epigenetic studies of twins can improve our understanding of how genetic, environmental and stochastic factors impact upon epigenetics, and how such studies can provide a comprehensive understanding of how epigenetic variation affects complex traits. The regulation of gene expression plays a pivotal role in complex phenotypes, and epigenetic mechanisms such as DNA methylation are essential to this process. The availability of next-generation sequencing technologies allows us to study epigenetic variation at an unprecedented level of resolution. Even so, our understanding of the underlying sources of epigenetic variability remains limited. Twin studies have played an essential role in estimating phenotypic heritability, and these now offer an opportunity to study epigenetic variation as a dynamic quantitative trait. High monozygotic twin discordance rates for common diseases suggest that unexplained environmental or epigenetic factors could be involved. Recent genome-wide epigenetic studies in disease-discordant monozygotic twins emphasize the power of this design to successfully identify epigenetic changes associated with complex traits. We describe how large-scale epigenetic studies of twins can improve our understanding of how genetic, environmental and stochastic factors impact upon epigenetics, and how such studies can provide a comprehensive understanding of how epigenetic variation affects complex traits. The term epigenetics was originally introduced to describe how interactions between genetics and environment can give rise to phenotypes during development [1Waddington C.H. The epigenotype.Endeavour. 1942; 1: 18-20Crossref Google Scholar]. Epigenetics today more specifically defines cellular modifications that can be heritable, but appear unrelated to DNA sequence changes, and can be modified by environmental stimuli [2Holliday R. Epigenetics: an overview.Dev. Genet. 1994; 15: 453-457Crossref Google Scholar, 3Russo V.E.A. et al.Epigenetic Mechanisms of Gene Regulation. Cold Spring Harbor Laboratory Press, 1996Google Scholar]. At present, epigenetic mechanisms typically comprise DNA methylation and histone modifications, but also include many other mechanisms such as ATP-based chromatin-remodeling complexes, Polycomb–Trithorax protein complexes, non-coding RNA mediated gene-silencing, and potentially prions, transcription-factor binding, and other mechanisms involved in generating and maintaining heritable chromatin structure and attachment to the nuclear matrix. Epigenetic mechanisms play an essential functional role in complex organisms as regulators of transcription. Central to epigenetic regulation is the modulation of chromatin structure, whereby the majority of epigenetic processes impact upon chromatin organization and maintenance. Next-generation sequencing technologies have been developed to assay epigenetic changes (Box 1) in high-throughput approaches, and high-resolution genome-wide epigenetic profiles promise a more complete understanding of the functional impact of epigenetics. Of these processes, DNA methylation is the mechanism that has been studied in the greatest depth, and we therefore focus predominantly on this mechanism in this review.Box 1Next-generation sequencing technologies applied to epigeneticsThe availability of next-generation sequencing technologies has recently allowed the survey of genome-wide epigenetic variation at high resolution [8Bock C. et al.Quantitative comparison of genome-wide DNA methylation mapping technologies.Nat. Biotechnol. 2010; 28: 1106-1114Crossref PubMed Scopus (225) Google Scholar, 99Harris J.R. et al.Sources of epigenetic variation at the MHC.Twin Res. Hum. Genet. 2010; 13: 262Google Scholar, 104Laird P.W. The power and the promise of DNA methylation markers.Nat. Rev. Cancer. 2003; 3: 253-266Crossref PubMed Google Scholar, 105Thu K.L. et al.Methylated DNA immunoprecipitation.J. Cell Physiol. 2009; 222: 522-531Google Scholar]. We describe some of these approaches briefly below. In addition, single-molecule sequencing technologies (e.g. [106Flusberg B.A. et al.Direct detection of DNA methylation during single-molecule, real-time sequencing.Nat. Methods. 2010; 7: 461-465Crossref PubMed Scopus (285) Google Scholar]) will probably reveal an even more complex and diverse layer of epigenetic mechanisms and modifications.DNA methylation(i)Bisulfite sequencing (Bi-seq) and reduced-representation Bi-seq (RRBS). These methods are based on bisulfite conversion of the DNA followed by sequencing. During bisulfite conversion, unmethylated cytosines are converted to uracils, whereas methylated cytosines remain unchanged, therefore complete bisulfite conversion is crucial to identifying correctly the unmethylated fraction of the genome. Bisulfite treatment cannot distinguish between methylated cytosines and 5-hydroxymethylated cytosines. Bisulfite-sequencing approaches survey DNA methylation across the entire genome (Bi-seq) or in a fraction of the genome (RRBS), which tends to be enriched for clusters of CpG dinucleotides.(ii)Methylated DNA immunoprecipitation sequencing (MeDIP-seq), methylated DNA capture by affinity purification sequencing (MeCAP-seq), and methylated DNA binding domain sequencing (MBD-seq). These techniques are based on extraction of the methylated sections of the genome by antibody or protein binding, followed by immunoprecipitation and sequencing. In some of these technologies the CpG density of a DNA fragment will affect precipitation, but algorithms that take this into account have been developed [9Down T.A. et al.A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis.Nat. Biotechnol. 2008; 26: 779-785Crossref PubMed Scopus (294) Google Scholar]. One advantage of the MeDIP-seq approach is the option to design antibodies specific to methylated cytosines, thereby allowing the separate detection of methylated cytosines and 5-hydroxymethylated cytosines.(iii)Methylation-sensitive restriction enzyme sequencing (MRE-seq). This approach detects typically unmethylated DNA and involves DNA digestion with methyl-sensitive restriction enzymes, such as HpaII, Hin6I and AciI, to select unmethylated DNA restriction enzyme cut-sites, followed by sequencing.Histone modificationChromatin immunoprecipitation followed by sequencing (ChIP-seq) is the standard approach used to detect histone-tail modifications.Non-coding RNAsLong non-coding RNAs can be detected using RNA-sequencing studies by selecting RNA transcripts that are not translated and are absent from protein databases, although the absence of protein products needs to be experimentally verified. Small non-coding RNAs, including miRNAs, typically require a separate library protocol and RNA sequencing experiment.Chromatin structure assaysRecent technologies have been designed to assay chromatin structure directly, and include DNase-seq, in which DNase I hypersensitivity (HS) sites are sequenced [107Boyle A.P. et al.High-resolution mapping and characterization of open chromatin across the genome.Cell. 2008; 132: 311-322Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar]; FAIRE-seq, or formaldehyde-assisted isolation of regulatory elements followed by sequencing, in which nucleosome-depleted DNA is isolated from human chromatin [108Gaulton K.J. et al.A map of open chromatin in human pancreatic islets.Nat. Genet. 2010; 42: 255-259Crossref PubMed Scopus (225) Google Scholar]; sono-seq, where sonication of cross-linked chromatin is followed by sequencing [109Auerbach R.K. et al.Mapping accessible chromatin regions using Sono-Seq.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14926-14931Crossref Scopus (89) Google Scholar]; 3C-seq, or chromosome conformation capture sequencing and related approaches [110Dekker J. et al.Capturing chromosome conformation.Science. 2002; 295: 1306-1311Crossref PubMed Scopus (1003) Google Scholar, 111Dostie J. et al.Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.Genome Res. 2006; 16: 1299-1309Crossref PubMed Scopus (342) Google Scholar, 112Simonis M. et al.Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C).Nat. Genet. 2006; 38: 1348-1354Crossref PubMed Scopus (475) Google Scholar, 113Tiwari V.K. et al.A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations.Genome Res. 2008; 18: 1171-1179Crossref Scopus (74) Google Scholar]; and nucleosome positioning assays. The availability of next-generation sequencing technologies has recently allowed the survey of genome-wide epigenetic variation at high resolution [8Bock C. et al.Quantitative comparison of genome-wide DNA methylation mapping technologies.Nat. Biotechnol. 2010; 28: 1106-1114Crossref PubMed Scopus (225) Google Scholar, 99Harris J.R. et al.Sources of epigenetic variation at the MHC.Twin Res. Hum. Genet. 2010; 13: 262Google Scholar, 104Laird P.W. The power and the promise of DNA methylation markers.Nat. Rev. Cancer. 2003; 3: 253-266Crossref PubMed Google Scholar, 105Thu K.L. et al.Methylated DNA immunoprecipitation.J. Cell Physiol. 2009; 222: 522-531Google Scholar]. We describe some of these approaches briefly below. In addition, single-molecule sequencing technologies (e.g. [106Flusberg B.A. et al.Direct detection of DNA methylation during single-molecule, real-time sequencing.Nat. Methods. 2010; 7: 461-465Crossref PubMed Scopus (285) Google Scholar]) will probably reveal an even more complex and diverse layer of epigenetic mechanisms and modifications. (i)Bisulfite sequencing (Bi-seq) and reduced-representation Bi-seq (RRBS). These methods are based on bisulfite conversion of the DNA followed by sequencing. During bisulfite conversion, unmethylated cytosines are converted to uracils, whereas methylated cytosines remain unchanged, therefore complete bisulfite conversion is crucial to identifying correctly the unmethylated fraction of the genome. Bisulfite treatment cannot distinguish between methylated cytosines and 5-hydroxymethylated cytosines. Bisulfite-sequencing approaches survey DNA methylation across the entire genome (Bi-seq) or in a fraction of the genome (RRBS), which tends to be enriched for clusters of CpG dinucleotides.(ii)Methylated DNA immunoprecipitation sequencing (MeDIP-seq), methylated DNA capture by affinity purification sequencing (MeCAP-seq), and methylated DNA binding domain sequencing (MBD-seq). These techniques are based on extraction of the methylated sections of the genome by antibody or protein binding, followed by immunoprecipitation and sequencing. In some of these technologies the CpG density of a DNA fragment will affect precipitation, but algorithms that take this into account have been developed [9Down T.A. et al.A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis.Nat. Biotechnol. 2008; 26: 779-785Crossref PubMed Scopus (294) Google Scholar]. One advantage of the MeDIP-seq approach is the option to design antibodies specific to methylated cytosines, thereby allowing the separate detection of methylated cytosines and 5-hydroxymethylated cytosines.(iii)Methylation-sensitive restriction enzyme sequencing (MRE-seq). This approach detects typically unmethylated DNA and involves DNA digestion with methyl-sensitive restriction enzymes, such as HpaII, Hin6I and AciI, to select unmethylated DNA restriction enzyme cut-sites, followed by sequencing. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the standard approach used to detect histone-tail modifications. Long non-coding RNAs can be detected using RNA-sequencing studies by selecting RNA transcripts that are not translated and are absent from protein databases, although the absence of protein products needs to be experimentally verified. Small non-coding RNAs, including miRNAs, typically require a separate library protocol and RNA sequencing experiment. Recent technologies have been designed to assay chromatin structure directly, and include DNase-seq, in which DNase I hypersensitivity (HS) sites are sequenced [107Boyle A.P. et al.High-resolution mapping and characterization of open chromatin across the genome.Cell. 2008; 132: 311-322Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar]; FAIRE-seq, or formaldehyde-assisted isolation of regulatory elements followed by sequencing, in which nucleosome-depleted DNA is isolated from human chromatin [108Gaulton K.J. et al.A map of open chromatin in human pancreatic islets.Nat. Genet. 2010; 42: 255-259Crossref PubMed Scopus (225) Google Scholar]; sono-seq, where sonication of cross-linked chromatin is followed by sequencing [109Auerbach R.K. et al.Mapping accessible chromatin regions using Sono-Seq.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14926-14931Crossref Scopus (89) Google Scholar]; 3C-seq, or chromosome conformation capture sequencing and related approaches [110Dekker J. et al.Capturing chromosome conformation.Science. 2002; 295: 1306-1311Crossref PubMed Scopus (1003) Google Scholar, 111Dostie J. et al.Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.Genome Res. 2006; 16: 1299-1309Crossref PubMed Scopus (342) Google Scholar, 112Simonis M. et al.Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C).Nat. Genet. 2006; 38: 1348-1354Crossref PubMed Scopus (475) Google Scholar, 113Tiwari V.K. et al.A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations.Genome Res. 2008; 18: 1171-1179Crossref Scopus (74) Google Scholar]; and nucleosome positioning assays. Epigenetic mechanisms are present in many taxa, but DNA methylation has been most extensively studied in mammals where it appears to be restricted to the cytosine base, and especially in the context of CpG dinucleotides. CpG dinucleotides are cytosine–phosphate–guanine sequences that typically cluster in genomic regions referred to as CpG islands (CGI), which are often located in gene promoters and exhibit low levels of DNA methylation. However, DNA methylation in mammals can also occur outside the CpG context, and this has been reported for example in embryonic stem cells [4Ramsahoye B.H. et al.Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5237-5242Crossref Scopus (439) Google Scholar]. Furthermore, although cytosine is typically methylated to 5-methylcytosine it can also be converted to 5-hydroxymethylcytosine, which could also play an important epigenetic role [5Kriaucionis S. 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Altogether, multiple factors including DNA sequence, DNA methylation, histone modifications, and other epigenetic and transcriptional activity factors contribute to chromatin regulation, which in turn modulates transcription and affects mammalian development and disease (Figure 1). Studies of twins have been crucial to disentangling the contribution of genetic factors to numerous complex traits. Twin studies in epigenetics have the potential to address two important questions. First, to what extent are epigenetic changes heritable and how much variation is there in epigenetic heritability across the genome? Comparisons within and between twin-pairs can help to determine the extent of epigenetic heritability and stability. Second, do epigenetic factors contribute to complex phenotypes? Monozygotic (MZ) twins are traditionally regarded as genetically identical, therefore any phenotypic differences within MZ twin pairs are classically attributed to environmental factors. However, epigenetic variants can also associate with phenotypic differences, and the identification and interpretation of such associations is currently an important area of research. Epigenetic studies of disease-discordant MZ twins, who are completely matched for genetics, age, sex, cohort effects, maternal influences and common environment, and are closely matched for other environmental factors, should be considerably more powerful in detecting disease-related epigenetic differences than epigenetic studies of unrelated disease cases and controls with different life-histories. In the following sections we consider the value of twin studies in epigenetics and discuss recent findings highlighting the possibility that epigenetic variation can be transmitted through generations and impact upon common diseases. Heritability is the proportion of the phenotypic variance in the population that is attributed to genetic variation. Heritability estimates are traditionally obtained by comparing the extent of similarity between relatives in classical twin studies, twin-adoption studies, sib/half-sib studies, and transgenerational family studies. Each has weaknesses, but for most traits twin studies are generally regarded as the most reliable because they are unbiased by age effects and offer the ability to separate common environment from genetic effects [33MacGregor A.J. et al.Twins. Novel uses to study complex traits and genetic diseases.Trends Genet. 2000; 16: 131-134Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 34Visscher P.M. et al.Heritability in the genomics era – concepts and misconceptions.Nat. Rev. Genet. 2008; 9: 255-266Crossref PubMed Scopus (407) Google Scholar]. In twins, heritability estimates compare concordance rates or intra-class correlations in monozygotic (MZ) and dizygotic (DZ) twins (Box 2). Twin comparisons of genome-wide epigenetic profiles can determine whether particular regions of the genome have higher epigenetic heritability estimates. In such regions, DNA methylation would appear to be influenced by genetic variation and DNA methylation variants would be relatively stable and could associate with genetic variants.Box 2Estimating genetic heritability using twin studiesHeritability refers to the proportion of the phenotypic variance in the population that is attributed to genetic variation, where genetic variants are represented either by additive genetic values (for narrow-sense heritability estimates, h2) or total genetic values (for broad-sense heritability estimates, H2). Twin heritability estimates usually refer to the narrow-sense heritability (h2), which is the proportion of the total phenotypic variance in the population that is attributable to additive genetic effects. Twin estimates of heritability compare phenotypic similarities in MZ and DZ twins, because MZ twins are genetically identical and share 100% of their germline sequence variation, whereas DZ twins are not genetically identical and share only on average 50% of germline sequence variants. In addition, MZ and DZ twins share the same uterus and birth date, and are exposed to very similar environmental factors in early development. In twins, h2 is typically calculated as twice the difference between MZ and DZ correlations,h2=2(rMZ−rDZ)where rMZ and rDZ are the correlations of the trait in MZ and DZ twins respectively. The advantage of the classical twin study is that it is possible to estimate and distinguish between the contributions of genetic, shared environmental, and unique environmental components to the phenotype. Heritability estimates are population-specific and, unless environmental factors remain constant, environment-specific. Interpreting heritability estimates should avoid common misconceptions [34Visscher P.M. et al.Heritability in the genomics era – concepts and misconceptions.Nat. Rev. Genet. 2008; 9: 255-266Crossref PubMed Scopus (407) Google Scholar]; for example, a large estimate of heritability does not necessarily r