PERSPECTIVES EPIGENETICS loci changing DNA methylation consistently OPINION in multiple individuals but also that these changes select for specific genes within Associating cellular epigenetic the genome that may have importance for disease risk or aetiology. Despite what sound like positive results models with human phenotypes of these EWAS approaches, we7,8 and others9,10 have noted that this typical EWAS Tuuli Lappalainen and John M. Greally approach suffers from multiple problems. Abstract | Epigenetic association studies have been carried out to test the These problems can be grouped into two categories: the robust identification of hypothesis that environmental perturbations trigger cellular reprogramming, with molecular (usually DNA methylation) downstream effects on cellular function and phenotypes. There have now been differences associated with the phenotype numerous studies of the potential molecular mediators of epigenetic changes by studied, and the interpretability of these epigenome-wide association studies (EWAS). However, a challenge for the field is results. The robustness issue is the typical the interpretation of the results obtained. We describe a second-generation EWAS focus of reviews addressing the problems of designing and carrying out EWAS7,9,10 approach, which focuses on the possible cellular models of epigenetic and usually includes discussion of cohort perturbations, studied by rigorous analysis and interpretation of genomic data. selection, statistical approaches used when Thus refocused, epigenetics research aligns with the field of functional genomics dealing with high‑dimensional data and to provide insights into environmental and genetic influences on phenotypic caution about the technical artefacts that 11–13 variation in humans. can occur in all genome‑wide studies . In this Opinion article, we focus instead on the issue of interpretability of even The first association of epigenetic processes described as epigenetic5,6 (BOX 1) and could the highest confidence DNA methylation with human disease occurred in the early thus be a molecular mediator propagating changes. Although our focus is on DNA 1980s when it was found that the DNA the memory of past cellular perturbations. methylation, as the most commonly studied of cancer cells had less 5‑methylcytosine Taking advantage of new assays to regulatory mechanism in human EWAS, the than normal counterpart cells1,2 and that interrogate increasing proportions of lessons apply to the broadening group of within this globally hypomethylated the genome, DNA methylation studies transcriptional regulators studied in human cancer genome the promoters of tumour were carried out on samples of cells from diseases. suppressor genes had paradoxically individuals affected by the phenotype of First, at the core of the interpretability increased DNA methylation3. The increased interest, comparing these profiles with concern are the often vague definitions DNA methylation of promoters of tumour comparable samples from individuals and terminologies used when discussing suppressor genes provided a reasonable lacking the phenotype. The genome-wide epigenetics, the biological hypothesis mechanism for silencing of those genes association study (GWAS) was used as being studied and the potential cellular and for their contribution to increased a model in these surveys, but instead of and molecular processes mediating the cellular growth. These observations raised looking at the relative allele frequencies phenotypic changes, as we discuss in the intriguing possibility that events other of DNA variants, these epigenome-wide detail below. Second, DNA methylation than DNA sequence mutations were association studies (EWAS) tested whether the can change in response to a diverse range occurring in cancer and caused cells to DNA methylation at individual or groups of of influences and does not solely reflect become chronically altered in terms of adjacent cytosines in the genome differed a chronic alteration of transcriptional transcriptional regulation. systematically in those with the phenotype regulation in response to a perturbation. The question then arose whether similar of interest. For example, a well‑recognized source cellular changes were taking place in disease EWAS has become a fast‑growing area of variability of DNA methylation is the phenotypes other than cancer. Of particular of research7. The typical results published presence of systematic differences in interest were those in which environmental for EWAS are those of significant changes cell‑subtype proportions between the groups exposures of various types were linked to in DNA methylation of modest degree, tested14,15. DNA sequence polymorphism is altered disease susceptibility, in particular substantially less than the potential also increasingly recognized to have a very those occurring a long time before the maximum change of 100%. It is generally powerful influence on transcriptional phenotype emerged. The observation that possible to infer associations between loci regulation, estimated to account for DNA methylation could be transmitted from changing DNA methylation and the likely a substantial proportion of differences of parent to daughter cells4 had prompted this functions of proteins encoded by nearby DNA methylation between individuals. transcriptional regulatory mechanism to be genes, indicating that not only are specific Twin studies show the heritability of DNA NATURE REVIEWS | GENETICS VOLUME 18 | JULY 2017 | 441 ©2017 Mac millan Publishers Li mited, part of Spri nger Nature. All ri ghts reserved. PERSPECTIVES Box 1 | A history of the word ‘epigenetics’ people who have already developed a phenotype. It has now been shown that DNA The adjective ‘epigenetic’ is derived from ‘epigenesis’, a theory of embryology that describes methylation in peripheral blood leukocytes specialized tissue structures developing from non-specialized precursors. Although the need for can be altered in response to increased such a theory is perhaps puzzling today, it was the counter to the preceding theory of body mass index25,26 or altered blood lipid ‘preformationism’, which instead proposed that development started with a miniature version 27 of the organism in a gamete. Aristotle (384–322 bc) was an early voice dissenting from the theory of profiles . Reverse causation occurring preformationism, having made observations of development in chickens that were inconsistent at the molecular level occurs when DNA with a preformationism process. With the development of microscopy in the 17th century, it was methylation is altered by transcription confirmed that mature organs were formed from structurally different precursors, referred to as an through the locus28,29, reflecting rather than emerging property of the developing organ, or epigenesis. causing the transcriptional differences In the 20th century, Waddington combined training in Drosophila melanogaster genetics (in the observed. laboratory of T. H. Morgan) and embryology, which involved microsurgical manipulations and grafting As a result of these issues, we have of embryos in tissue culture. His focus was on the regulators of cellular differentiation during proposed that no EWAS to date, which development, attempting to understand how cells changed their differentiation fates, responding to includes our own studies, can be said to inductive effects evoked by humoral factors acting on cells with different competence to respond. be fully interpretable8. The goal of this He described these effects in terms of what he called the epigenetic landscape67,98, depicted in terms of a hillside within which a valley branches out into a continuously bifurcating delta of valleys, down article is to focus on how this situation which a ball could be imagined to roll, representing cell differentiation and distinctive fates (FIG. 1a). can be improved and how the concerns of Waddington’s goal was to bridge the gap between the two fields of embryology and genetics99, vaguely defined terminology and biological with the downhill slope the process of epigenesis and the delta of valleys of cell fates formed because hypotheses, confounding factors and of the influence of genes. His goal to reconcile the embryologists’ model of epigenesis and the ambiguous causation can be addressed. potential role of genetic influences in development was reflected by his describing the cell fate Although we focus on humans and on landscape as epigenetic, fusing the words epigenesis and genetic. diseases other than cancer, and on the Waddington’s idea was re‑interpreted in 1958 by Nanney, who made the assumption that analysis of DNA methylation rather than epigenetic systems were those responsible for cellular memory, whether differentiation in differences in gene expression or chromatin a vertebrate cell or the memory of a prior exposure to galactoside by Escherichia coli33. In the states between cases and controls, many of 1970s, this idea of cellular memory as an epigenetic property of cells was taken a step further by both Riggs5 and Holliday6, who noted that DNA methylation offered a potential transcriptional these insights should apply more broadly. regulatory mechanism that could be maintained through cell division. The further re‑interpretation We propose that, although we currently by Riggs and Holliday described epigenetic properties more in terms of Nanney’s definition face substantial challenges in generating focused on cellular memory and less attributable to Waddington’s model reconciling