From stem cells to the law courts: DNA methylation, the forensic epigenome and the possibility of a biosocial archive

The growth in epigenetics continues to attract considerable cross-disciplinary interest, apparently representing an opportunity to move beyond genomics towards the goal of understanding phenotypic variability from molecular through organismal to the societal level. The epigenome may also harbour useful information about life-time exposures (measured or unmeasured) irrespective of their influence on health or disease, creating the potential for a person-specific biosocial archive. Furthermore, such data may prove of use in providing identifying information, providing the possibility of a future forensic epigenome. The mechanisms involved in ensuring that environmentally induced epigenetic changes perpetuate across the life course remain unclear. Here we propose that a potential role of adult stem cells in maintaining epigenetic states provides a useful basis for formulating such epidemiologically-relevant concepts. Epigenetics encompasses different mechanisms of gene expression regulation, the most commonly discussed ones being DNA methylation, histone modifications and non-coding RNAs.1,2 The epigenetic mechanism most studied to date is DNA methylation—addition of a methyl group (CH3) at the 5’ position of a cytosine base, typically at CpG dinucleotides which are often clustered in CpG-rich DNA segments called CpG islands.3,4 This chemical addition is made by a covalent bond and is stable over time. Since epigenetic processes are believed to be modifiable by environmental (i.e. non-genetic) factors, much of the current interest in epigenetics lies in understanding how the environment influences gene expression—even though, as currently analysable, epigenetics is unlikely to hold all of the answers for this broad and rather complex question.2,5 Technological advances now allow epigenome-wide investigations to be performed in large human populations at affordable costs—especially with respect to DNA methylation.6,7 Although other measures are technically possible, they remain prohibitively expensive on a population scale. Epidemiological studies involving DNA methylation pertain to one of the most recent branches of epidemiology—epigenetic epidemiology.8 Large-scale profiling of DNA methylation levels has been applied to case-control studies,9,10 densely-phenotyped cohorts11 and in some instances serial samples from the same longitudinal cohort.12 This allows investigation of the determinants of variation in DNA methylation and their importance in the context of different health outcomes and traits, as evidenced by the steady rise in epigenetic epidemiology publications (Figure 1) and the numerous studies reported in this issue of the IJE.13–24 Ten year publication trajectory in epigenetic epidemiology (2005-2014). Methylation modifications are relatively stable. Indeed, such stability is important in the maintenance of methylation modifications that regulate developmental processes, such as cell differentiation.25–28 On the other hand, methylation modifications are sometimes reversible, and there is evidence that demethylation plays an important role in biological processes.29–31. Stochastic processes also contribute to some of the plasticity observed in the methylome. The short-term responsiveness of the epigenome to environmental challenges is an area that has not been widely studied. Experimental studies of short-term exposure to particulate air matter have demonstrated that DNA methylation changes occur within hours of exposure.32 Inference can also be made with respect to responsiveness to exposures in utero detected at birth, given the specific time window of exposure.48 Critical windows of exposure can however be difficult to determine in many instances, as the epigenetic measure inevitably captures a cross-sectional measure of epigenetic differences as opposed to being contemporaneous with the timing of the exposure itself. Long-term effects of an early-life exposure on methylation levels might mediate at least some of the associations between exposures and phenotypes at later life stages, although scepticism needs to be maintained in the breadth of claims regarding DNA cytosine methylation, given it plays no important role in the development of such important model organisms as Caenorhabditis elegans,33,Drosophila melanogasterand Saccharomyces cerevisiae34 (a conclusion not altered by detection of very low levels of cytosine methylation in Drosophila35 and adenine methylation in both Drosophila and C. elegans36).It is commonplace in epidemiological studies to see claims that epigenetic processes may provide biological plausibility to potentially causal associations, which then represent potential targets for intervention.2,5,8,37–39 Epigenetic mediation through persistent changes induced in early life has indeed become a widely accepted hypothetical model for developmental programming,38–40 particularly in generic reviews of this field.39 This has motivated many studies, including examples published in this issue of the IJE, involving: early-life determinants of methylation levels shortly after birth16,17,20 and in adulthood14; longitudinal associations of methylation levels with neurocognitive function and behaviour in children18 and with physical and cognitive fitness in the elderly;23 and the potential mediating role of DNA methylation in the association between maternal smoking and birth weight.15 It should be noted, however, that epigenetic persistence is not a prerequisite for a programmed effect, since a transient change in DNA methylation could, in theory, set other biological processes in train which then precipitate long-term effects. Outside of the developmental programming literature there are also examples of long-term health effects of an exposure potentially mediated by DNA methylation. Lung cancer risk is higher among past smokers compared with never smokers, and the relative risk is maintained over time after quitting smoking.41 It is possible that long-term epigenetic modifications are mediators of this association, since past smoking has recently been associated with DNA methylation levels even decades after cessation42 (and could be considered a very useful biomarker of exposure at the tissue level). Nevertheless, disentangling mediation from other association-driving mechanisms—such as confounding and reverse causation—requires careful consideration,2,5,8,37 and statistical approaches to such mediation analysis are severely compromised by measurement error.43–46 Furthermore, the tissue from which methylation data are generated—blood, in the case of the above-referenced paper on dynamic methylation changes in relation to quitting smoking41 —is not a plausible candidate for a mediator between smoking and long-term, post-cessation, risk of lung cancer. These serious problems are unlikely to restrain over-confident claims of causality and mediation in the reporting of studies incorporating methylation measures, however. Perusal of existing literature indicates that evidence of long-term effects of exposures on DNA methylation is largely limited to assumptions of persistence made in studies where methylation is measured at a single time point and related to historical exposure data. This approach is, of course, limited in the inferences that can be made, although access to serial samples from the same individuals with prospectively collected exposure data can help to improve this. Longitudinal data sets are valuable in many epidemiological contexts. Repeated measures of epigenetic signatures allow the modelling of change in methylation over time in tandem with single or repeated measures of an exposure that occurs prior to the methylation measurement.47 One can assess the persistence of differential methylation observed at birth across the life course, whether any changes are reversible and what factors may explain reversibility. For example, an epigenome-wide association study identified that methylation levels in seven gene regions were associated with maternal smoking during pregnancy. Four of these remained associated with maternal smoking throughout childhood and adolescence.48 Furthermore, models can be developed and tested to evaluate the intensity, duration and timing of an exposure on DNA methylation.48 The extent to which methylation data can indicate the particular timing of exposures—for example, during the intrauterine period or during puberty—is currently poorly understood. A DNA methylation score derived from smoking-responsive DNA methylation sites has previously been used as an indicator of smoking status, i.e. to categorize current, former and never smokers49 and the widely used ‘epigenetic clock’ has been used to predict age from methylation patterns.23,50 The use of the ‘epigenetic clock’ as an indicator of biological rather than chronological age is increasingly being mooted, based on the assumption that DNA methylation signatures provide an index of cellular ageing (as in the case of telomere length). The potential utility of DNA methylation in forensics is beginning to generate interest, to the extent that by 2009 a popular prime-time TV programme, Law and Order: Special Victims Unit ran an episode entitled ‘Perverted’, in which DNA methylation analysis was utilized to demonstrate that DNA at a crime scene had been generated in vitro (and was unmethylated rather than the in vivo methylated copy) and thus appeared to have been planted.51 Although it was a far-fetched storyline, the methods alluded to in the programme had been reported in Forensic Science International: Genetics.52 The body fluid or tissue source of DNA obtained at crime scenes can also be of forensic importance, and DNA methylation can help in this identification.53,54 Other potential forensic uses include distinguishing between a monozygotic (MZ) twin pair to establish which twin left a DNA sample at a crime scene. Methylation patterns, unlike the germ-line genome, can be identifying due to the phenotypic information that they reflect55,56 (although potentially, somatic mutations detected in complete high-coverage genome sequencing could also identify a particular MZ twin). This is not entirely of purely theoretical interest, as identical twins have indeed gone unprosecuted in such situations.57 Between-twin methylation differences appear stable enough to be useful even when there is a substantial time interval between when a sample from one twin was recovered from a crime scene and the twin pair had samples collected and examined.58 Indeed, if it transpires that there is any non-germ-line genetic variation-based paternal-to-offspring transmission of methylation, as some epigenetic enthusiasts claim, this could even be used in paternity tests involving an offspring of one of a pair of male MZ twins. Transgenerational epigenetic inheritance has been reviewed in detail59 but remains a contentious area of epigenetic research, in particular with respect to the public health importance of epigenetic variations transmitted across generations.60 Despite overblown claims regarding potential forensic uses for DNA-based face-shape prediction,61 common genetic variation cannot provide much useful information about individual characteristics beyond sex and (probabilistically and problematically) ethnicity and related characteristics including eye, hair and skin colour,62–64 known collectively as ‘externally visible characteristics’, or EVCs.65 DNA methylation offers the possibility of moving beyond conventional EVCs and adding identification of other aspects of the bodily habitus of the (generally unwitting) source of forensic blood (or other tissue) samples. The epigenetic clock, mentioned above, is producing mean absolute differences of chronological and estimated age of only 3–4 years in adults,50 which be useful for the of could have been the source of recovered samples. have been tested within a forensics with of to long-term blood sample and to DNA above, smoking behaviour is a that can be DNA methylation with of current from being has also been and useful based on DNA methylation data may be but evidence is limited to generate these at For other aspects of such as body the of genetic and epigenetic data can improve over the use of genetic data but this not approach the of being useful for of smoking and could help in the identification of recovered samples. other DNA methylation data could in point to both identifying characteristics and to For example, there has been considerable interest in potential epigenetic effects of and other although this is limited to studies at maternal during could on the offspring epigenome that have forensic as as the epidemiological discussed alluded to above, maternal smoking during such identifying and in some persistent offspring DNA methylation providing of this is also evidence that maternal or other during is associated with changes to the This has than theoretical interest, since in the states have one case to as through which have in that could their during can be to The role of epigenetic understanding in formulating evidence as to in maternal during can influence development and outcomes has been discussed in the beyond DNA methylation data could be used to establish that such have been during a particular pregnancy. addition to maternal use during has also been to methylation changes in although this is not as methylation of other maternal largely in the context of model studies, are currently to Indeed, the beyond the to which it should that to effects on the offspring through transmitted epigenetic changes could also become a for the from potential of the of one societal the very same persistent epigenetic that allow for a potential forensic epigenome can also in the context of epidemiological studies, to the of the of exposures to which individuals are to as the The potential and of exposure across the life course, during which and biological processes are on the the possibility of a biosocial archive. The to including exposures samples collected offers a of considerable to as the health effects of smoking as an for the birth of smoking has been the most widely exposure in epigenetic as discussed methylation of the from and other even after statistical for reported smoking This indicates that the methylation measure provides a indicator of long-term exposure to smoking than of the a conclusion by the that the associations of reported smoking behaviour with were for methylation. 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