Nearly 50% of adults in the United States have experienced a mood or anxiety disorder at some point in their lives. Mood and anxiety disorders are among the most debilitating diseases worldwide—the World Health Organization’s latest Global Burden of Disease report ranked depression among the top three most prevalent causes of disability globally, accounting for the highest single proportion of years lived with disability around the world 1 2 . Panic disorder, as well as self-inflicted injury, which is often precipitated by depression, were also high on the WHO list 2 . Mood, anxiety disorders and self-inflicted injury are also profoundly expensive, imposing high direct and indirect costs on individuals, industry, and health systems, alike 3 .

The search for causes of common mood and anxiety disorders and suicide has spanned at least a century of research in a wide range of disciplines. However, a gap remains between studies focused on exogenous environmental determinants such as negative life events 4 or neighborhood social context 5 6 and studies focused on genetic determinants and biological correlates such as abnormalities in brain circuitry 7 . The recent growth in interest in epigenetic studies in human populations has been fueled, in part, by the potential of epigenetics to bridge this divide 8 .

Epigenetic modifications are those reversible, mitotically heritable alterations in genomic expression that occur independent of changes in gene sequence 9 . Rather they occur via methylation of DNA or alterations to chromatin structure that either impede or facilitate access to the DNA by transcription factors and associated complexes 9 . Epigenetic modification of expression has been demonstrated to mediate the interplay between environmental stimuli and physiologic—and pathophysiologic—change throughout the life course 10 . Early research has been promising 11 , demonstrating epigenetic involvement in the pathophysiology of depression 12 , anxiety disorders 13 , and suicide 14 . However, a central challenge to understanding the role of epigenetic modifications in psychopathology is limited access to brain tissue in extant studies, where inference has been limited to assessments of epigenetic modification of peripheral tissue samples which may not be related to pathophysiologic processes underlying psychopathology.

Given the growing interest in behavioral epigenetics it appears to be the appropriate time to take stock, and to synthesize the peer-reviewed literature about epigenetic modification in the etiology of common mood and anxiety disorder and suicide. In reviewing the existing literature, we aimed to summarize the state of the science, identify methodological challenges, and offer potential solutions to address these challenges.

We systematically reviewed the literature about epigenetic factors in the etiology of mood and anxiety disorders and suicide. We restricted our search to English-language, peer-reviewed articles. Our review encompassed the literature published between January 1^st, 2001 and December 1^st, 2011—we limited our review to the years following the sequencing of the human genome to reflect current thinking about the genetics of psychopathology. The literature reviewed here was identified via the MEDLINE and PSYCHINFO databases.

Our original search yielded 1273 articles, 600 of which were judged to consider epigenetic factors in the etiology of mood and anxiety disorders and/or suicide after screening by title. Another 368 were discarded after screening by abstract because they did not consider epigenetic mechanisms or disease outcomes of interest. Of the remaining 232, articles were included in the review if they fulfilled the following criteria:

· Included original data about a n > 1 sample of human subjects

· Used DSM-III, DSM-IV, or ICD-10 criteria to classify participants as having common mood or anxiety disorders (including and limited to PTSD, GAD, Phobias, Panic Disorder, or Depression) OR used coroners’ reports to classify cause of death by self-inflicted injury

· Objectively assessed epigenetic mechanisms relating to the etiology of mood and anxiety disorders or suicide

After reading the complete manuscripts, 211 were excluded because they did not meet our criteria, above. This left 21 articles from the original search considered in this review. Reference lists from these articles were searched, and yielded no further articles which fulfilled the inclusion criteria, yielding a final total of 21 articles reviewed here. Figure1 shows a flow diagram of our search strategy.

Because of the diversity of outcomes in which we were interested, the genetic pathways considered in the extant literature, and the methods used to both assess for epigenetic mechanisms and statistically analyze the findings, a meta-analysis of the results would not have been appropriate or feasible. For each of the 21 papers, we extracted the following information: the outcome of interest; sample population; proportion male; proportion White; number of cases and controls; loci considered; tissues sampled; laboratory techniques used; statistical analyses employed; and summary of the findings.

Table 1 includes a detailed review of each of the studies included in our review. The majority (12) of the studies included in the literature considered epigenetic modification in the etiology of suicide 14 15 16 17 18 19 20 21 22 23 24 25 . Five studies considered epigenetic factors and mood disorders (all five were concerned with depression) 12 26 27 28 29 , and the remaining four studies considered epigenetic factors in the etiology of anxiety disorders (three considered PTSD and one considered panic disorder) 13 30 31 32 .

*N/I; not indicated.

All of the studies concerned with epigenetic factors in suicide assessed gene expression and methylation profiles in the post-mortem brains of suicide completers relative to non-suicide controls 14 15 16 17 18 19 20 21 22 23 24 25 , although one study also included adjuvant data about methylation profiles in peripheral leukocytes 17 . Among studies regarding epigenetic factors in the etiology of depression, one study assessed methylation and expression profiles in the post-mortem brain 27 , another assessed methylation and expression of tissue in buccal cells 28 , and the remaining three assessed methylation and expression profiles in peripheral blood 12 26 29 . All four studies about epigenetic mechanisms in anxiety disorders assessed methylation and expression in whole blood 13 30 31 32 .

We systematically reviewed the peer-reviewed literature about the role of epigenetic modification in the etiology of common mood and anxiety disorders and suicide. Twenty-one papers were published between 2001, the publication of the human genome project, and 2011. The majority (12) of studies we found were concerned with evidence of epigenetic changes in the post-mortem brains of suicide completers, with other studies considering epigenetic factors in the etiology of depression, PTSD, and panic disorder. A plurality focused on epigenetic regulation of genes involved in amine, glucocorticoid, and serotonin metabolism in the production of common mood and anxiety disorders and suicide; studies also considered epigenetic modification of a diverse array of other genes.

Given the small number of studies, drawing substantive conclusions about how epigenetic modifications in specific genes may be operating in the etiology of the diseases in question is not possible at this stage. Our review occasions a synthesis of methodological limitations of the extant literature and recommendations on how investigators may best approach this area in future studies.

Five methodological limitations to this literature emerge from our review. The first is that studies in this area have suffered from small sample sizes, the consequences of which include lack of power and increased false discovery rates. Second, existing studies have been limited to assessing epigenetic modification in the post-mortem brain or the peripheral blood following disease diagnosis, and drawing inference from either tissue type is problematic. Third, studies have used different techniques to assess epigenetic modifications that may produce heterogeneous results. Fourth, few studies have assessed environmental antecedents to epigenetic modifications in extant studies. Fifth, there appears to be little consensus regarding genome-wide vs. candidate-gene approaches.

The first methodological limitation to this literature is the use of small sample sizes in most studies, a ubiquitous problem in molecular epidemiology 33 . Of the studies we reviewed, only one included more than fifty cases (e.g., subjects with the outcome). Compounding small sample sizes in studies overall, many of the studies we reviewed limited epigenetic analysis to a subset of the total study population. Small sample sizes limit study power, therefore increasing the likelihood of type II error (e.g., the proportion of false negative findings) 33 . More dangerously, underpowered studies also increase the “false discovery rate” or the number of significant findings that fall into type I error (e.g., the proportion of false positive findings), as demonstrated in Equation 1 34 35 36 .

F D R = α 1 − p r i o r α 1 − p r i o r + p o w e r ∗ p r i o r

^ “prior” indicates the proportion of tested hypotheses that are actually correct.

In this equation, the false discovery rate (FDR) is inversely proportional to power (1-beta), such that low power also yields high FDR, driving up Type I error. Therefore, given the small sample sizes employed in the majority of studies we reviewed, it is likely that the findings suffer from high proportions of both type I and type II error.

A second limitation is the use post-mortem brain or peripheral cell tissues for epigenetic analyses. Seven of the 21 studies we reviewed analyzed epigenetic modification in peripheral blood cells, and one study analyzed epigenetic modification in buccal mucosa. Although all human cells carry the full endowment of genetic material, cells modify gene expression to efficiently carry out their diverse functions as they specialize, silencing some genes while activating others in line with their physiologic responsibilities. Epigenetic modification is the physiologic process by which genes are silenced or primed for expression 37 38 . The pathophysiology of mood-anxiety disorders and suicide is localized to the brain and it remains therefore unclear how gene expression in peripheral tissues correlates with physiologically meaningful gene expression in the brain. However, even epigenetic studies using post-mortem brain tissue have challenges. Three of the 21 studies we reviewed analyzed post-mortem brain tissue. While these studies assessed epigenetic changes in the appropriate organ, assessing post-mortem brain tissue carries its own challenges. This is problematic with respect to temporality between exposure and outcome, because post-mortem brains, by definition, can only be harvested after death, and therefore epigenetic modification can only be ascertained after the occurrence of the outcome. Moreover, death often involves acidosis, which may contribute to the instability of genetic material 39 40 41 , increasing the likelihood of misclassifying epigenetic modification and increasing the chances of spurious findings. Therefore, much more work is needed to help us understand the physiologic significance of both peripheral tissue and brain methylation patterns.

A third limitation to the literature is that published studies used different laboratory techniques to measure the degree of epigenetic modification. With respect to DNA methylation alone, there are a number of gene-specific assays currently in use, including bisulfite reaction based DNA sequencing methods, which include bisulfite genomic sequencing PCR 42 and/or methylation specific PCR 43 ; genome-wide screens, such as CpG island microarrays 44 and Restriction Landmark Genomic Scanning for Methylation (RLGS-M) techniques 45 ; and methylated DNA immunoprecipitation (MeDIP) 46 . There are few studies that have compared the sensitivity and specificity of each method, although a recent study compared two bisulfite sequence-based assays (which are very similar) head-to-head and found as much as an 18% difference in identification of methylated CpG islands in biological replicates of human embryonic stem cells 47 . To our knowledge, there are no “gold standard” assays for most epigenetic markers. Therefore, differential use of assays may present a source of misclassification bias in studies, which would ultimately increase the rate of type II error rate in the extant literature.

Fourth, studies in our review largely failed to assess the environmental exposures thought to produce epigenetic change to begin with. Only three out of 21 of the studies reviewed here included any assessment of a common environmental stressor with respect to epigenetic modifications and their relationship with common mood and anxiety disorders and suicide 14 19 20 . This is an important limitation, as there is ample data demonstrating the importance of environmental stressors in the etiology of these disorders 48 . Without assessing common environmental stressors antecedent to epigenetic modifications, our studies fail to adequately test dominant hypotheses about the mediating role of epigenetic changes between environments and outcomes in common mood and anxiety disorders and suicide.

The fifth limitation is the lack of consensus regarding genome-wide vs. candidate-gene approaches in epigenetic studies. Three of the studies we reviewed used genome-wide approaches 12 30 32 , while the remaining 18 studies assessed for epigenetic modifications of candidate genes. Both approaches have limitations. With respect to genome-wide studies, the analyses (and findings) are often unfocused. Unlike in genome-wide association studies, there is no agreed upon method for analysis and synthesis of data or for adjustment for multiple comparisons in genome-wide epigenetic studies. In particular, appropriate adjustment for multiple comparisons can be problematic, increasing the proportion of false-positive findings 49 . Candidate-gene approaches benefit from being hypothesis-driven, and therefore more amenable to thoughtful, model-based study designs. However, candidate-gene approaches face their own limitations. Candidate-gene studies are more likely to yield overall negative results, as these studies test only one hypothesis, as compared to genome-wide studies which test more global hypotheses about the role of epigenetic modification anywhere in the genome influencing risk for outcomes of interest. As candidate-gene approaches are more likely to yield negative overall findings, there is a high probability of publication bias, whereby the literature about candidate-gene epigenetic modification is likely to be highly enriched for positive findings 35 .

Research into the epigenetic mechanisms that may underlie common mood and anxiety disorders and suicide has the potential to unite the heretofore disparate bodies of work that have characterized the pathophysiology of these disorders and their population causes, respectively. However, at this nascent stage in the development of this literature, there are several methodological challenges, discussed above, that have yet to be addressed. With respect to these challenges moving forward, studies about the role of epigenetic regulation in the etiology of common mood and anxiety disorders can improve in the following ways.

As sufficient power and small sizes have dogged extant studies in this area, future studies require larger sample sizes, maximizing power and minimizing the false discovery rate. Furthermore, systematic comparisons of assays used to assess epigenetic modifications are needed as is consensus around what constitute ‘gold standard’ laboratory techniques for assessing epigenetic modification. Another challenge to this literature is the validity of using epigenetic data from peripheral blood, epithelial tissue, or post-mortem brain specimens. However, given the obvious limitations to sampling brain tissue in representative populations of living subjects, sampling these tissues may, in the long-term, remain the best available option. Future work associating epigenetic modifications with changes in gene expression and correlating epigenetic modifications in peripheral tissues with brain function may offer one way to address these limitations. Moreover, work in neuroscience to characterize the relationship between real-time brain imaging studies and epigenetic modification in brain regions sampled among patients undergoing neurosurgery, when living-patient brain samples can be collected, could yield imaging markers of epigenetic regulation in the brain. These approaches represent a handful of the many lines of inquiry that could improve our capacity to assess epigenetic modification in the brain. Future studies in this area would also do well to measure epigenetic modification in relation to its environmental antecedents so as to better assess dominant hypotheses about the mediation of the relationship between environmental exposures and common mood and anxiety disorders and suicide by epigenetic modification.

Lastly, both genome-wide and candidate gene approaches have their role in epigenetic analyses. Genome-wide approaches may be more appropriate for exploratory analyses 50 . However, candidate gene approaches may be better suited for hypothesis-testing regarding the roles of individual genes or sets of genes hypothesized to function in a particular way in disease etiology. It is important that investigators in this area are attuned to the particular strengths and weakness of each approach so that each is used appropriately in studies about the role of epigenetics in the etiology of common mood and anxiety disorders and suicide.

The authors declare that they have no competing interests.

AME extracted data from studies and drafted the manuscript. MRH carried out the literature search and edited the manuscript for intellectual content. SG edited the manuscript for intellectual content. KCK conceived the review, specified review inclusion and exclusion criteria, and edited the manuscript for intellectual content. All authors have read and approve the final version.