What do DNA methylation studies tell us about depression? A systematic review

There has been a limited number of systematic reviews conducted to summarize the overview of the relationship between DNA methylation and depression, and to critically appraise the roles of major study characteristics in the accuracy of study findings. This systematic review aims to critically appraise the impact of study characteristics on the association between DNA methylation and depression, and summarize the overview of this association. Electronic databases and gray literatures until December 2017 were searched for English-language studies with standard diagnostic criteria of depression. A total of 67 studies were included in this review along with a summary of their study characteristics. We grouped the findings into etiological and treatment studies. Majority of these selected studies were recently published and from developed countries. Whole blood samples were the most studied common tissues. Bisulfite conversion, along with pyrosequencing, was widely used to test the DNA methylation level across all the studies. High heterogeneity existed among the studies in terms of experimental and statistical methodologies and study designs. As recommended by the Cochrane guideline, a systematic review without meta-analysis should be undertaken. This review has, in general, found that DNA methylation modifications were associated with depression. Subgroup analyses showed that most studies found BDNF and SLC6A4 hypermethylations to be associated with MDD or depression in general. In contrast, studies on NR3C1, OXTR, and other genes, which were tested by only few studies, reported mixed findings. More longitudinal studies using standardized experimental and laboratory methodologies are needed in future studies to enable more systematical comparisons and quantitative synthesis.


Introduction
A number of systematic reviews on susceptible genes and gene-environment interplay provide a comprehensive list of putative genetic and environmental risk factors for depression [1][2][3][4][5][6] . In contrast, there has been little compilation of our knowledge of DNA methylation modifications and depression.
To our knowledge, there are five reviews, including only one systematic review so far on the relationship between DNA methylation and depression [7][8][9][10][11] . Generally, they suggested that altered DNA methylations may be associated with the etiology of depression. Lockwood et al. in their narrative review of epigenetic findings in both animal and human studies concluded that epigenetics could play an important role in depression and suicide in humans 7 . Again, Uddin et al 8 ., using a similar approach, studied the role of sex in DNA methylation and posttraumatic stress disorder and major depressive disorder (MDD), and suggested that sex differences in DNA methylation among those genes known to influence brain development may explain the sexually dimorphic risk for developing post-traumatic stress disorder and MDD. Another narrative review found the inverse association between adverse environmental factors, i.e., early-life stress, and the epigenetic modification of gene expression 9 . A review examined the association between DNA methylation of seven candidate genes and depression, and found that brain-derived neurotropic factor (BDNF) and nuclear receptor subfamily 3 group C member 1 (NR3C1) gene methylation levels may be related to depression, whereas the relationship between serotonin transporter gene (SLC6A4; synonyms: 5-HTT and SERT) and depression was inconsistent 11 . One recent systematic review assessed both animal and human studies and identified the correlation between burnout/depression and global and candidate-gene DNA methylation 10 . However, this review did not examine the influence of experimental and statistical methodologies and analyses on findings.
Although a few reviews are published to explore the relationships between DNA methylation modifications and depression, there has been no review critically examining experimental methodologies and verification of laboratory testing in humans. The experimental methodologies and laboratory testing are closely linked with the accuracy of results. In addition, these reviews only focused on some aspects; for example, exploring the roles of sex and stress in this relationship. In this review, we aimed to (1) systematically synthesize the major findings on DNA methylation and depression, (2) compare the similarities and differences across different studies, including experimental and laboratory factors and statistical analyses used, which might partially explain some inconsistencies in the results, and (3) discuss the challenges and opportunities for future studies.

Materials and methods
The processing and reporting of the results of this systematic review were guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 2009 revision 12 . To ensure a thorough and systematic review of the literature, two methods were used to retrieve all the studies on relevant topics. We conducted a search of the computerized bibliographic databases PubMed, Web of Science, EMBASE, Medline, and Cochrane Library. The search strategy is detailed in Supplementary Appendix 1. The literature search comprised articles published until December 2017. A snowball technique was then applied to identify further studies. In addition, we manually searched other resources for other relevant studies. The reference lists of selected articles, review articles on relevant topics, and the gray literatures were screened. Figure 1 presents the process of study selection.
All suitable articles were evaluated with regard to their internal validity based on the four selection criteria as follows: (1) if they used a clear diagnosis criteria for depression (e.g., depression in general, major depressive disorder, depressive symptoms, or other types of depression), specifically the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) and its updates 13 , and the International Statistical Classification of Diseases and Related Health Problems, 10th revision (ICD-10) 14 or other generally accepted diagnostic criteria; (2) if they examined the association between DNA methylation and depression; and (3) if they provided a statistical indicator (i.e., coefficient) or original data to estimate the relationship between DNA methylation and depression. Articles were excluded if (1) they did not specify a clear diagnosis for major depression, major depressive disorder, unipolar depression, or other types of depression or (2) they were not written in English.
Two authors (M. Li and X. Li) independently screened all the retrieved articles. Inconsistencies in interpretation were resolved through group discussions (X. Li, M. Li, and X. Meng). Endnote and RefWorks were the bibliographic softwares used. Data on author(s), year of publication, sample size, study design, study cohort, experimental methods, type of tissues, candidate genes or genome, DNA purification method, DNA methylation method, DNA methylation validation, genotyping, gene expression, experimental factors, statistical methods, and major findings were extracted independently. For those studies with multiple reports, a single record denoted one study with the information extracted from multiple reports. Group discussions dealt with all the inconsistent interpretations. The reviewers endeavored to contact the original authors of the studies for any missing information in order to gather complete and consistent study information. Open-ended questions were used to prevent misleading answers.
Because of the divergence of candidate genes and genomes, for example, some studies used the candidate-gene approach and others examined the whole genome, we grouped the summarized findings according to the number of studies available, including etiological studies and treatment studies. The etiological studies were then further divided into the following subgroups, including (1) BDNF, (2) SLC6A4, (3) NR3C1, (4) oxytocin receptor (OXTR), (5) other genes, and (6) genome-wide. Some articles were involved in multiple separate analyses as their data permitted.

Results
A total of 67 articles met our eligibility criteria. Figure 1 shows the detailed information of the process of study selection. Table 1 presents a summary of study characteristics of these selected studies. Supplementary Appendix 2 provides a list of the references for all the selected articles corresponding to their order in Table 1. Most of the reviewed articles were published between 2014 and 2017, especially in the past 4 years. The selected studies mainly focused on adults and seniors (58/67), covering a total number of 11,935 subjects worldwide (North America: 18/67, Asia: 21/67, Europe: 24/67, and Australia: 6/67). We also evaluated study quality, including design (study design, sample size, and subject characteristics), implementation (biological sample, DNA methylation method, purification of DNA extraction, and validation of methylation), analysis (analytical method, batch effect, genotyping, and gene expression), and interpretation of results. Most studies in this review were case-control with hospital-or general population-based cohorts. There was a wide variety in terms of sample size, ranging from 11 to 1024. Whole blood was the most commonly used biological sample analyzed by generally accepted DNA methylation methods, such as bisulfite conversion with pyrosequencing. Both parametric and non-parametric statistics were used. Importantly, most of these studies did not analyze the influence of batch effect on their results (64/67), except the three studies targeted on genome-wide variations.
This review was designed to apply evidence-based approaches to summarize the findings between DNA methylation and depression. High heterogeneity was identified among the studies reviewed. The Cochrane guidelines do not recommend using quantitative methods, such as meta-analysis, to synthesize the research findings. Thus, qualitative methods were then used to summarize  Etiological studies: genome-wide Although all genome-wide studies found significant methylation modifications associated with depression, both hyper-and hypomethylation correlations were reported. Inconsistent results were also noted. For instance, in one study, hypermethylation was previously found in a pilot study, but was not present on its replication 15 ; a significant decrease in mean methylation was observed among females, but not among males 16 ; lower methylation levels were found among severe MDD patients vs healthy controls, but no difference between severe vs remitted patients 17 ; and one study found both hypermethylations in some processes (e.g., brain development and tryptophan metabolism), and hypomethylations in other tissues (e.g., lipoprotein) 18 . Generally, sample sizes were not associated with study designs or major findings. However, studies with large sample sizes were more likely to use DNA purification methods and examine gene expression than those with smaller samples. Results from studies with large sample sizes are considered to be more reliable.

Etiological studies: candidate-gene
Generally, most studies found BDNF and SLC6A4 hypermethylation to be associated with MDD or depression. Studies on NR3C1, OXTR, and the rest of candidate genes, which were tested by only a few studies, reported mixed findings (hyper-and hypomethylation modifications and non-significant differences). The promoter regions and CpG islands were frequently targeted in these studies. The sample size in each group varied dramatically from 12 to 1024. Some of these studies also had gene expression for significant findings. Replications of findings were better in BDNF and SLC6A4 than in other studied genes. Studies with a longitudinal study design, reliable laboratory arrays, and statistical analyses were more likely to provide robust results.

Treatment studies
Findings in this group are more inconsistent compared to those in etiological studies. Half of the studies did not identify any significant methylation sites associated with antidepressant responses, and the rest had mixed significant findings (hyper-and hypomethylations) on different candidate genes. Again, the promoter regions and CpG islands were the major targets. This group of studies had a higher level of heterogeneity compared to other subgroups, as treatment history and stages of treatments may influence methylation modifications.

Discussion
This review firstly explored the role of DNA methylation in depression considering both the laboratory and analytic factors that could potentially confound the findings. A total of 67 articles were included in this review. The majority of the selected studies were recently published and were from developed countries. Whole blood was the most common tissue used in these analyses. Bisulfite conversion, along with pyrosequencing, was widely used to test DNA methylation level. There was a high heterogeneity among the studies in terms of the laboratory and statistical methodologies used and study designs. Large sample size and laboratory verification (DNA purification and DNA methylation validation) are the major characteristics important for accurate results.
The findings of our study are as follows.
(1) For studies using candidate-gene approaches, BDNF, NR3C1, SLC6A4, and OXTR genes were the most frequently studied genes. Promoters and CpG islands were the common targeted regions. Overall, most of the studies found that BDNF and SLC6A4 hypermethylations were associated with depression. Studies on NR3C1, OXTR, and other candidate genes reported mixed findings in terms of methylation modification and depression. Again, promoters and CpG islands still were the focus. (2) All genome-wide studies found significant methylation sites, including hyper-and hypomethylations. (3) For studies that explored antidepressant treatment responses, their results were inconsistent as they targeted on a number of different genes and different stages of treatment. (4) Large-sample size studies were more likely to use DNA purification methods, examine gene expression in their analyses, and provide more reliable results.
ZBTB20 exists in the hippocampal neurons and cerebellum granule cells 25 , and plays a role in many processes, including neurogenesis, glucose homeostasis, and postnatal growth 26 . It may also have an impact on the development and regionalization of the human hippocampus, which has been found to be related to depression [27][28][29] .
Both HP1BP3 and TTC9B are linked to estrogen signaling. HP1BP3 is highly expressed in the brain and is related to a number of physical and behavioral phenotypes in mice, such as dwarfism, impaired bone mass, impaired maternal behavior, and anxiety 30,31 . Lower HP1BP3 has been found to be associated with postpartum depression and Alzheimer's disease in humans 21,32 . TTC9B has been identified to be related to gonadal hormones 33 and may be linked to hippocampal synaptic plasticity, which is critical for hippocampal long-term potentiation and depression 34 . These markers in peripheral blood may indicate estrogenmediated epigenetic changes in the hippocampus and in turn, potentially, raise the vulnerable phenotypes based on their actions in brain 21 .
The GRIN2A gene provides the instructions for making a protein called glutamate receptor subunit epsilon-1 in human encoded GluN2A, which is one of the components (subunit) of a subset of N-methyl-D-aspartate (NMDA) receptors. They are involved in normal brain development and are responsible for changes in the brain in response to experience (synaptic plasticity), learning, and memory 26 . Methylation modifications in GRIN2A may play a key role in determining the function of NMDA receptors. Generally, gene promoter-region methylation could repress the gene expression, but the methylation on gene body can be positively correlated with expression activity 35 . This suggests that the hypermethylation of the GRIN2A gene body may result in the overexpression of NR2A and, thus, promote vulnerability for MDD via up-regulating NMDA receptor-dependent glutamatergic signaling 36 .
Hypomethylations were also observed among depression patients on the following genes: WD repeat domain 26 (WDR26), the promoter region of miRNA4646, 5hydroxymethylcytosine (5-hmc), and 5-methylcytosine (5mc) 17,23,[37][38][39][40][41] . Consistent with our findings on WDR26, previous studies have found that the hypomethylation of WDR26 in depressed individuals may be related to lower gene-expression levels 42 . Additionally, the decreased blood transcription levels of WDR26 were associated with depression-related phenotypes [42][43][44][45] . 5-mc is a methylated form of the DNA base cytosine, which could be involved in the regulation of gene transcription. Its presence is important for the maintenance of the active chromatic state and for neurogenesis at non-promoter CpG islands 46 , and is associated with stable and long-term transcriptional silencing of promoters 47 . 5-mc is also found to be involved in the critical mechanism mediating genomic imprinting. This process has been identified as a key for normal development, and its abnormal imprinting can result in disorders such as Prader-Willi, Angelman, and Beckwith-Wiedemann syndrome 47 .
5-hmc is a product of conversion of 5-mc. It is related to the regulation of gene expression, prompting DNA demethylation. The three ten-eleven translocation (TET) enzymes oxidize each step in the demethylation of 5-mc. 5-mc is first converted to 5-hmc, then to 5-formylcytosine (5fC), and then to 5-carboxylcytosine (5caC), each by TET1-3 48 . Reduced levels of TET1 and, subsequently, 5hmc cause impaired self-renewal of stem cells 49 .
Notably, inconsistent results were identified within the same studies among different subgroups; for example, different sexes 16 , processes (e.g., brain development, tryptophan metabolism, and lipoprotein) 18 , tissues (white blood cells, brain, and sperm) 50 , or between pilot and replication studies 15 .

Findings on etiological candidate-gene studies
For candidate-gene studies, the majority (11/12) of studies on BDNF found BDNF hypermethylation were associated with cases suffering from depression. Most of the studies had relatively large sample sizes and examined DNA purification. This is consistent with the recent reviews on BDNF and depression. Chen et al. indicated that more than half of the studies showed an increased BDNF methylation in depressed patients. Bakusic et al. concluded in their review that hypermethylation was consistently found in MDD subjects across the three studies selected 10 . The BDNF gene provides the instructions for making a protein found in the brain and spinal cord, and promotes the survival of nerve cells (neurons). It is actively involved in the growth, maturation, and maintenance of these neurons, and in the regulation of synaptic plasticity, which is important for learning and memory 26,51 . It is reported that changes in the methylation level of the BDNF promoter are associated with its lower expression in the prefrontal cortex 52 and its activity in the hippocampus in animal studies 53 . A similar decrease in BDNF levels was also found in the serum and plasma of MDD patients; thus, it is hypothesized that MDD is related to impaired neuronal plasticity 53 .
Positive associations between SLC6A4 methylation modifications and depression have also been identified in many studies in this review and previous reviews 10, 11 . All longitudinal studies in this review and studies with more comprehensive considerations of lab and statistical work have consistently found that depression patients had SLC6A4 hypermethylation compared to controls. SLC6A4 gives the instructions for making a protein in the brain that is involved in the regulation of serotonergic signaling by transporting serotonin or 5-hydroxytryptamine (5-HT) from synaptic spaces into presynaptic neurons 54 and in the regulation of emotional behaviors 55 . The alterations of SLC6A4 play an important role in brain development and function in humans 56 . It has been hypothesized that DNA hypermethylation may result in the reduction of SLC6A4 expression and 5-HT reuptake, which in turn may increase the vulnerability to affective disorders at critical stages of development 57,58 .
Findings on NR3C1, OXTR, and other genes were less coherent. Both hypo-and hypermethylation levels were noted in depressive patients compared to controls. No significant associations between DNA methylation on these genes and depression were also reported by some studies. Similar findings were also found by recent reviews 10,11 . NR3C1 is the receptor to which cortisol and glucocorticoids bind. It regulates gene transcriptions and is linked to development, metabolism, and immune response 59,60 . OXTR is a receptor of the hormone and neurotransmitter oxytocin 61,62 . It presents in the central nervous system and plays an important role in modulating various behaviors, such as stress and anxiety, social memory and recognition, sexual and aggressive behaviors, bonding/affiliation, and maternal behavior [63][64][65] . We found that some of the selected studies had certain limitations in terms of the type of study design, sample size, and range of laboratory work and statistical analyses. Due to the high heterogeneity across the selected studies, this review could not provide more conclusive results on these genes in terms of relationships between DNA methylation modifications on these genes and depression.

Findings on treatment studies
Findings of this subgroup were less consistent than those of the other two subgroups analyzed. However, this is in line with another recent review on DNA methylation, and clinical response to antidepressants in MDD patients was inadequate to provide any consistent support for such a relationship 66 . Both the increased and decreased DNA methylation levels on SLC6A4 and BDNF genes were associated with the use of antidepressant medications, whereas MAOA methylation modification was not linked to antidepressant response. The relationship between antidepressant treatment and DNA methylation of certain genes has been reported, i.e., BDNF DNA methylation modification was associated with decreased gene expression, which can lead to MDD 67 . The use of antidepressants can restore the decreased BDNF to the normal level and alleviate depressive symptoms 53,67 . Inconsistencies across all these findings may be explained by different ethnicities, duration of treatments, and pharmacogenetic heterogeneities 68,69 . Investigations on antidepressant response should cover all the different treatment stages, since the level of DNA methylation may be altered during the treatment 70 .

Strengths and limitations
This review synthesizes the findings on DNA methylation associated with depression and critically appraised the major study characteristics that can significantly impact this association, including study design, study population, targeted genetic variations, methylation arrays, types of tissues, DNA purification, methylation validation, appropriate statistical methods, and the consideration of downstream analyses, e.g., genotyping and gene expression.
However, there are several limitations to be noted. First, this review was designed to provide an overview of the relationship between DNA methylation and depression. Therefore, all eligible studies with a wide range of genomic coverage, i.e., targeted genes or whole genome, and different types of study designs were included. As many study characteristics were heterogeneous, no pooled results were made to simply estimate this relationship. Second, although we used subgroup analyses to synthesize homogeneous studies, different types of tissues, study designs, phenotypes of the outcome, comparison groups, analytic methods, and sample sizes can still lead to inconsistent results. Third, most of studies were crosssectional. DNA methylation level is dynamic and potentially reversible, and can be affected by a number of environmental factors. Findings from these crosssectional studies may not be able to reveal the true nature of this complex relationship. Finally, only English databases were searched, which may limit the comprehensiveness of eligible studies.
Overall, we found that hyper-and hypomethylations on promoter regions and CpG islands of a number of genes were significantly associated with the disease. Most of the studies applied the widely acceptable laboratory techniques and statistical analyses, which made the pooled results more likely to reach a consistent finding. Future studies should adopt longitudinal study designs to explore the dynamic change of methylation levels. To allow for a systematic comparison of studies, there should be an agreement upon the consistent set of standards involving a minimum set for the items for the execution and reporting of methylation studies similar to what is required for the reporting of clinical trials, systematic reviews and meta-analysis 12,71 . Gene expression should also be routinely added into the research to uncover how, when, and what underlying mechanisms link these identified methylation sites to depression. This would advance the field and provide a firm base for the evidence on the relationship between DNA methylation and depression.
prepared the draft of this manuscript. X.M. and C.D. designed this review. T.Z. and R.J. assisted with the interpretation of the results. X.M. oversaw the project, provided feedback on all steps of the search, data extraction, and interpretation. All authors contributed to the writing and editing of the manuscript.