Accepted Manuscript Title: SLC6A4 methylation as an epigenetic marker of life adversity exposures in humans: A systematic review of literature Author: Livio Provenzi Roberto Giorda Silvana Beri Rosario Montirosso PII: DOI: Reference:
S0149-7634(16)30139-7 http://dx.doi.org/doi:10.1016/j.neubiorev.2016.08.021 NBR 2567
To appear in: Received date: Revised date: Accepted date:
10-3-2016 18-7-2016 23-8-2016
Please cite this article as: Provenzi, Livio, Giorda, Roberto, Beri, Silvana, Montirosso, Rosario, SLC6A4 methylation as an epigenetic marker of life adversity exposures in humans: A systematic review of literature.Neuroscience and Biobehavioral Reviews http://dx.doi.org/10.1016/j.neubiorev.2016.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
SLC6A4 methylation as an epigenetic marker of life adversity exposures in humans: A systematic review of literature
Livio Provenzi1*, Roberto Giorda2, Silvana Beri2, Rosario Montirosso1
1
0-3 Center for the at-Risk Infant, Scientific Institute IRCCS Eugenio Medea, Bosisio
Parini, LC, Italy 2
Molecular Biology Lab, Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, LC,
Italy
* Corresponding author: Livio Provenzi, 0-3 Center for the at-Risk Infant, Scientific Institute IRCCS Eugenio Medea, via Don Luigi Monza 20, 23842 Bosisio Parini, LC, Italy. Telephone: +39-031-877464. E-mail:
[email protected]
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HIGHLIGHTS
DNA methylation epigenetically modifies gene transcription in response to adversities
Altered DNA methylation associates to adverse socio-emotional development
The serotonin transporter gene (SLC6A4) regulates socio-emotional development
Evidence on human SLC6A4 methylation in response to adversities is reviewed
Altered SLC6A4 methylation emerged as a potential marker of adversity exposure
ABSTRACT The application of epigenetics to the study of behavioral and socio-emotional development in humans has revealed that DNA methylation could be a potential marker of adversity exposure and long-lasting programming of health and disease. The serotonin transporter gene (SLC6A4) is a stress-related gene which has well-documented implications for behavioral and socio-emotional development and which has been shown to be susceptible to transcriptional regulation via epigenetic mechanisms. In the present paper, a systematic review of papers assessing the association among adversity exposures, SLC6A4 methylation and developmental outcomes is reported. Nineteen studies were included. Findings revealed that SLC6A4 methylation has been investigated in humans in association with a number of prenatal and postnatal adverse exposures, encompassing maternal depression during pregnancy, perinatal stress exposure, childhood trauma and abuse, and environmental stress. SLC6A4 is confirmed as a relevant biomarker of early adversity exposures, and epigenetic mechanisms occurring at this gene appear to play a critical role for programming. Nonetheless, specific methodological issues still need to be addressed in future human behavioral epigenetic research.
Keywords: early adversity; epigenetics; methylation; serotonin transporter; SLC6A4; stress; systematic review 2
1. INTRODUCTION Emerging evidence suggests that early exposure to adverse environmental events is linked to altered socio-emotional development and stress susceptibility (Griffiths and Hunter, 2014). Nonetheless, there is an extraordinary variability in the degree of individual differences in stress vulnerability and resilience. Animal studies and human literature suggest that epigenetic processes—modifying DNA transcriptional functionality without changing DNA sequence—might be candidate mechanisms for the embedding of life adversities into the organism’s biology, behavior and brain functioning. Moreover, there is considerable evidence that the serotonin (5-HT) system mediates stress susceptibility (Lesch, 2011). Recent research suggests that epigenetic processes might be involved in 5-HT modulation through changes of the transcriptional function of the gene encoding for the serotonin transporter (5HTT), namely the SLC6A4 gene (Booij et al., 2013). In the current paper, we present a systematic review to assess SLC6A4 epigenetic dynamics in association with the exposure to a set of adverse experiences in humans. Preliminarily, we provide a theoretical background for epigenetics and its application to human behavioral and socio-emotional development (namely, human behavioral epigenetics). 2. DNA METHYLATION AS AN EPIGENETIC STRESS MARKER 2.1.
Epigenetics and DNA methylation
Epigenetics refers to functional changes in the DNA or associated proteins without structural modifications of the DNA sequence itself (Jaenisch and Bird, 2003). In other words, whereas the genome concerns the potential genetic information contained in the DNA which informs gene transcription and expression, the epigenome defines which genes of this potential repertoire are actually expressed (Booij et al., 2013).
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Epigenetic mechanisms encompass DNA methylation, modification of histones and microRNA (miRNA). DNA methylation is a covalent modification occurring at the level of DNA dinucleotides rich in cytosine and guanine (CpG sites, where the “p” indicates the phosphate group within the dinucleotide pair) through the addition of a methyl group onto the cytosine ring of the CpG dinucleotide (Szyf, 2009). The modification of histones consists in the remodeling of chromatin, the structure in which the DNA is wrapped up, and involves covalent modifications at the level of histone tails in order to promote an open or closed chromatin structure. Open chromatin associates with the accessibility to transcriptional factors and increased rates of gene expression, whereas closed chromatin structures are linked to inhibited transcription. Finally, miRNAs have recently been suggested to play a different role in epigenetic regulation, maintaining or actively changing the transcription of genes in a heritable way (Szyf, 2009). Here, we focused on DNA methylation as the most studied epigenetic mechanism involved in environmental modifications of the 5-HT system and its outcomes in human development. 2.2.
DNA methylation and transcriptional activity
DNA methylation is fostered by DNA methyltransferase enzymes (DNMTs), which are responsible for the binding of the methyl group from donor S-adenosyl-methionine onto the 5’ position of the CpG dinucleotide. Different DNMTs are responsible for the de novo appearance of methylation patterns (DNMT3; Okano et al., 1999) and for the maintenance of methylation patterns across cell differentiation and replication (DNMT1; Hermann et al., 2004). DNA methylation occurring at the level of regions relevant for gene regulation and expression, characterized by a greater density of CpG sites (i.e., CpG islands), are of great concern for researchers and clinicians. These regions include exons, promoter regions and enhancers (Hyman, 2009). DNA methylation occurring at these regions affects gene 4
expression and the availability of specific proteins within the central nervous system, both directly and indirectly. On the one hand, the binding of a methyl group to the transcriptionally relevant CpG site of a given gene associates with inhibited transcriptional activity and gene silencing. On the other hand, the different epigenetic mechanisms are thought to interact to produce patterns of increased or reduced transcriptional sensitivity. For example, higher methylation in both exonic and intronic regions is effective in recruiting histone deacetylases, leading to gene silencing (Jones et al., 1998; Nan et al., 1998). Incidentally, a high density of methylation at the level of CpG islands within coding-relevant regions is capable of silencing gene expression and leads to a loss of protein production, which could have similar behavioral and neurological consequences as those observed in the case of genetic alterations involving polymorphisms and mutation (Meaney and Szyf, 2005). 2.3.
Behavioral epigenetics
Behavioral epigenetics refers to “the application of principles of epigenetics to the study of physiological, genetic, environmental, and developmental mechanisms of behavior in human and non-human animals” (Lester et al., 2011, p. 14). More specifically, behavioral epigenetics concerns the behavioral outcomes and antecedents of epigenetic modifications, including DNA methylation. As such, behavioral epigenetics studies how genes—and gene expression patterns, in particular—are affected by environmental stimuli and contribute to the programming of health and disease later in life (Lester et al., 2016). Lester and colleagues (2011) provided a preliminary map of behavioral epigenetic studies to date, highlighting that the main topics of investigation encompassed substance abuse (Maze and Russo, 2010), learning and memory (Gutierrez-Mecinas et al., 2011; Miller and Sweatt, 2007), neurodevelopment (Lesseur et al., 2014), parenting effects (Meaney and Szyf, 2005), psychiatric illness (Tsankova et al., 2007), and, more generally, stress exposure (Stankiewicz 5
et al., 2013). From 2011 to date, behavioral epigenetics has accumulated an exponentially increasing number of research outputs, with a pivotal interest in the associations among early adversities, epigenetic changes (e.g., DNA methylation) and neuro-behavioral (Lester et al., 2015; Stroud et al., 2016), physiological (Conradt et al., 2016; Oberlander et al., 2008), and socio-emotional (Booij et al., 2013; Nikolova et al., 2014) stress-related outcomes in human infants, children and adults (Griffiths and Hunter, 2014; Montirosso and Provenzi, 2015). The present review focuses on stress-related outcomes, with particular attention to DNA methylation occurring at the level of SLC6A4 as the primary gene involved in the regulation of the serotoninergic system and a key regulator of stress susceptibility (Gaspar et al., 2003; Lesch, 2011). 3. THE 5-HT SYSTEM ROLE IN SOCIO-EMOTIONAL DEVELOPMENT AND STRESS REGULATION The serotonergic neurotransmission is known to affect a wide range of developmental outcomes, including behavior, cognition, emotion and stress regulation (Hood et al., 2006). In animals, serotonergic projections to the periaqueductal grey region have been shown to play a critical role in the suppression of behavioral and biological responses to challenging environmental conditions (Mohammad-Zadeh et al., 2008). In humans, 5-HT is located both in the central nervous system and in peripheral tissues. In the brain, it is located in the neurons of the median and dorsal raphe nuclei, in the cerebral cortex, and in the hippocampus (Torres et al., 2003). Peripherally, 5-HT can be found in the gastrointestinal tract as well as in lungs, placenta, blood, platelets, and lymphocytes (Mohammad-Zadeh et al., 2008; Torres et al., 2003). Importantly, amygdala, hypothalamus, and the pituitary adrenal gland, which are deeply involved in the onset, duration and regulation of socio-emotional stress regulation, are densely innervated by serotonin neurons (Hood et al., 2006). 6
3.1.
The serotonin transporter: Genetic variations regulating the serotonergic
system A key regulator of the serotonergic system is the serotonin transporter (5-HTT), which removes the 5-HT released in the synaptic cleft. The 5-HTT is produced by a specific gene, namely SLC6A4 (see Figure 1), which is widely expressed in the central nervous system (Lesch, 2011). The expression of SLC6A4 is regulated by several mechanisms, including genetic variations. Among them, 5-HTTLPR (Heils et al., 1995) is a polymorphism that has been extensively studied in association with stress-related personality traits, behavior, biological and physiological correlates in humans (Canli and Lesch, 2007). A handful of studies suggest that the 5-HTTLPR is a candidate polymorphism for socio-emotional stress susceptibility (Lesch, 2011; Montirosso et al., 2015; Pauli-Pott, Friedl, Hinney, & Hebebrand, 2009; Pluess et al., 2011). The 5-HTTLPR can be individually expressed in short (S) or long (L) allelic variants, with the former being linked to reduced 5-HTT transcription and heightened risk for negative developmental outcomes, such as emotional and affective disorders in adolescence and adulthood (Lesch, 2011).
- Figure 1 about here -
Since the mid-1990s, genetic studies on 5-HTTLPR in humans have reported that the genetically determined variability of 5-HTT availability has a number of phenotypic correlates. For example, individuals with at least one S allele (i.e., S-carriers) have been found to have higher scores in neuroticism, a personality trait associated with anxiety and depression in human adults (Lesch et al., 1996). Notwithstanding, inconsistencies have been reported in subsequent research, with large cohort studies failing to replicate the same significant 7
associations (Sirota et al., 1999). Such discrepancies might be at least partially ascribed to specific sample composition in different studies, the low effect size generally obtained between a single gene polymorphic variation and complex behavioral traits, gene-to-gene interactions, and the role of environmental encounters in exacerbating or reducing the susceptibility conveyed by the genotype. Trying to obviate the issue related to the low effect size for the association between 5HTTLPR and a complex personality trait such as neuroticism, other researchers moved toward the study of gene-to-endophenotype associations (Meyer-Lindenberg and Weinberger, 2006). Endophenotype has the advantage of depicting simpler and biologically-relevant outcomes of genetic variations, which might be closer to the level at which a gene operates. Compared to more remote phenotypic outcomes, such as observed behaviors and self-rated temperament or personality, the endophenotype has a higher biological plausibility. As such, it was expected to reveal less inconsistency. Indeed, a low resolution electromagnetic tomography (LORETA) study initially reported an association between 5-HTTLPR and prefrontal cortex limbic excitability in response to a cognitive task in laboratory: S-carriers had a higher activity in the anterior cingulate cortex compared to non-carriers (Fallgatter et al., 2002). Other studies, using functional magnetic resonance imaging (fMRI), documented associations between the presence of the S allele and greater amygdala activation during emotion-eliciting tasks (Hariri et al., 2002; Pezawas et al., 2005). Nonetheless, further investigations led to an inconsistent picture of the association between 5-HTTLPR and cerebral functioning. Some studies suggested a right-lateralized effect of the S allele (Bertolino et al., 2005), whereas others documented a bilateral effect (Canli et al., 2006). In sum, research on personality-based, behavioral, and neuro-imaging correlates of 5HTT genetically determined variability suggested that a far more complex picture was 8
needed. The stress vulnerability conveyed by the 5-HTTLPR variation probably reflects developmental biochemical mechanisms that act on the structural connectivity and functional interactions within neural circuits that regulate emotional regulation in individuals exposed to early adverse experiences. The genetically determined variability conveyed by the 5-HTTLPR was not sufficient to account univocally for behavioral variability, which led to a new wave of studies focused on the interactions between 5-HTTLPR and the environment (i.e., Gene x Environment, or G x E studies). 3.2.
Embedding the serotonin transporter gene into the environment: G x E
studies Animal studies on the interaction between 5-HTTLPR and one’s environment documented that a stressful condition, such as maternal separation during the first months of life, resulted in deficient social adaptation and reduced skills of social cognition and interaction in rhesus monkeys (Bennett et al., 2002). Nonetheless, these difficulties were much more evident when the monkeys were carrying the S allele of 5-HTTLPR. In humans, carriers of the S allele are up to twice as likely to get depressed after the exposure to stressful events, such as bereavement and maltreatment (Caspi et al., 2003). In human infants, research has documented that stressful experiences might be better understood through a focus on the interplay between 5-HTTLPR variations and environmental influences (Barry et al., 2008). For example, an interaction between maternal anxiety and infants’ 5-HTTLPR genotype was found in 6-month-old infants with negative emotionality, as rated through parental reports (Pluess et al., 2011). The 5-HTTLPR x E interaction was also found on negative emotionality observed during a stressful daily care procedure at 12 months (Pauli-Pott et al., 2009). Maternal interactive behavior at 7 months and infants’ 5-HTTLPR behavior were jointly predictive of attachment security evaluated at 15 months (Barry et al., 9
2008). More specifically, despite the fact that the S allele was found to convey a higher risk for emotional disorders, S-carrier infants benefited from maternal behavioral support more than their L-homozygous counterpart. A recent study using a well-established procedure to test socio-emotional stress regulation in young infants (i.e., the Face-to-Face Still-Face, FFSF paradigm; Tronick et al., 1978) documented higher negative emotionality in 4-month-old Scarrier infants (Montirosso et al., 2015). Nonetheless, maternal social engagement behaviors were found to moderate this association, lowering negative emotionality in genetically at-risk S-carrier infants, but not in genetically protected L-homozygous infants. 3.3.
A new concept of G x E interactions for the 5-HT system: DNA methylation
The evidence of an association between serotonin-related genetically-conveyed vulnerability (e.g., the S allele of the 5-HTTLPR polymorphism) and early adversities has recently underscored the need for a better comprehension of the mechanisms through which early adversity “gets under the skin” and is embedded in the developing biology of a human infant and child (Hyman, 2009). As previously mentioned (Canli and Lesch, 2007), G x E studies have contributed to identify specific correlates of interactions that affect emotional behaviors and socio-emotional stress vulnerability. Nonetheless, further studies are still needed to understand the epigenetic mechanisms involved in SLC6A4 transcription in association with exposure to adverse environments. During recent years, evidence suggesting that epigenetic mechanisms (mainly, altered patterns of DNA methylation) might explain how a set of different early adverse events might lead to less-than-optimal socioemotional stress regulation and to further-in-life health and disease has begun to accumulate. According to the behavioral epigenetics view, environmental encounters might interact with genotype affecting the organization and structure of DNA chromatin (Champagne and Curley, 2009). Thus, gene expression is sensitive to environmental influences through 10
epigenetic mechanisms, processes that alter the accessibility of the coding portions of a given gene to molecular transcriptional agents and lead to permanent alterations in gene expression (Weaver et al., 2004). Consistently, DNA methylation variations at the level of specific stress-related genes have been associated with early adversity exposures in human subjects (Griffiths and Hunter, 2014). In turn, the altered pattern of gene expression and protein synthesis leads to changes in cell functioning in the target tissue and contributes to the emergent phenotype (Hyman, 2009). 3.4.
Preclinical studies on the association between SLC6A4 and serotonin
transporter expression In human lymphoblast cell lines, higher SLC6A4 methylation has been associated with reduced mRNA levels (Philibert et al., 2007). This finding suggests that it is plausible to link the differences in SLC6A4 promoter region methylation with variations in the serotonin transporter availability via altered gene expression. In other words, DNA methylation occurring at the promoter region of the SLC6A4 is actually capable of regulating gene expression. This association has been replicated in vitro by Olsson and colleagues (2010), showing that both compete and partial promoter methylation of the SLC6A4 significantly reduces gene expression. In rhesus monkeys, early adverse exposures have been associated with minor expression of the serotonin transporter gene and with behavioral disinhibition during stressful laboratory sessions (Kinnally et al., 2008; 2009). Notably, lower peripheral blood mononuclear cells have been documented in association with another downregulator of the SLC6A4 expression, namely the 5-HTTLPR S-homozygous allelic form (Kinnally et al., 2010). Notwithstanding, not all the studies concurrently demonstrated a significant and negative association between greater methylation and lower expression of the serotonin transporter gene. Vijayendran and colleagues (2012) did not report this association, 11
despite SLC6A4 expression was assessed in peripheral tissues, but not in central tissues, as is commonplace in humans (Uebelhack et al., 2006). The authors concluded that, if a growing number of studies demonstrates that adversity-related changes in SLC6A4 methylation associate with adverse developmental outcomes in humans, these effects might be more visible in central nervous system splices of the SLC6A4. 4. THE PRESENT STUDY We have reviewed studies assessing the relationship between early adverse events and SLC6A4 methylation in human subjects aged 0 to 18 years. First, as the structure of SLC6A4 is complex, the principal goal was to determine which SLC6A4 gene regions have already been assessed for adversity-related methylation changes. Indeed, different sections of the gene might be differentially involved in 5-HTT expression, so the region at which methylation occurs might or might not convey behaviorally observable consequences for socio-emotional development and stress response (Vijayendran et al., 2012). A second objective was to individuate specific adverse events that have been associated with SLC6A4 methylation and their consequences at the level of socio-emotional functioning and stress response. Finally, on the basis of the findings, a picture of the state of the art for human SLC6A4 behavioral epigenetics is provided, highlighting the main discoveries and limitations and addressing further directions for clinically relevant epigenetic research. 5. METHODS A computer-based literature search was conducted in three main databases: Scopus, ISI Web of Science, and PubMed. The title/abstract search string was “methylation” AND “SLC6A4.” The search string was purposively non-discriminative in order to include a great number of studies in the first round, to be further controlled through critical appraisal.
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- Figure 2 about here -
Figure 2 is a flow chart of the systematic review, and it highlights the step-by-step criteria for screening and the record count (excluded and included results at each step). Records were considered eligible if they were already published or available as online previews ahead of print. No previous reviews, theoretical pieces, viewpoints and letters were included. Other exclusion criteria are listed in Figure 2 notes. A total of 19 studies were included in the present review (see Table 1).
- Table 1 about here -
6. RESULTS – PART 1: SLC6A4 METHYLATION INFORMATION 6.1.
Tissues
The main source for the SLC6A4 methylation analysis was peripheral blood (14 out of 19 studies). In three studies, researchers obtained both peripheral blood from the mother and cord blood at birth (Devlin et al., 2010; Montirosso et al., 2016; Provenzi et al., 2015). Buccal cells (Ouellet-Morin et al., 2013) or saliva (Chau et al., 2014) were each reported once. 6.2.
SLC6A4 regions
Regions analyzed in the papers included in the present review are reported in Figure 3. The number of CpG sites assessed for methylation status widely varied among the studies, ranging from 2 (Koenen et al., 2011) to 83 (Alexander et al., 2014; Wankerl et al., 2014). One study did not report the exact number of CpG sites, reporting only 11 CpG units (van der Knaap et al., 2015).
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6.3.
SLC6A4 unit of analysis
Nearly all of the studies adopted a CpG site-specific level of analysis (17 out of 19). The assessment of the overall region methylation was carried in about half of the studies (10 out of 19). In order to limit the bias of multiple testing, some researchers adopted principal component analysis (PCA; 4 out of 19 studies) or factor analysis (FA; Duman and Canli, 2015). 7. RESULTS – PART 2: SAMPLE CHARACTERISTICS AND STUDY DESIGN The sample size varied widely among the studies, ranging from 25 (Wang et al., 2012) to 939 subjects (van der Knaap et al., 2015). The majority of the papers reported on methylation in adult human subjects (15 out of 19), whereas three studies (Devlin et al., 2010; Montirosso et al., 2016; Provenzi et al., 2015) provided data on newborns and Chau and collaborators (2014) studied on school-age children. One study investigated DNA methylation in a cohort of adolescents (van der Knaap et al., 2015). The majority of the studies were retrospective in their nature (14 out of 19), apart from Devlin et al. (2010), Provenzi et al. (2015) and Montirosso et al. (2016). Alasaari and colleagues (2012) as well as Chau and collaborators (2014) adopted a cross-sectional study design, assessing both adverse exposure and methylation during adulthood and childhood, respectively. 8. RESULTS – PART 3: ADVERSE EVENT INFORMATION A wide range of adverse events was covered, including prenatal exposure to maternal depression, perinatal pain-related stress exposure, childhood traumas encompassing sexual and physical abuse, peer-related trauma, and environmental stress. 8.1.
Prenatal adverse exposures Devlin and colleagues (2010) reported the association between exposure to maternal
depression and antidepressant treatment during pregnancy and the methylation status of 14
SLC6A4 in both mothers and newborns. Maternal mood was measured at two time points: at mid-pregnancy (Time 1) and at 33 weeks of gestation (Time 2). Methylation was measured at 10 CpG sites upstream of the SLC6A4 transcriptional start site (TSS). The methylation status of the maternal SLC6A4 promoter was significantly lower in mothers with higher depression symptoms at Time 1 at six out of ten CpG sites. Maternal SLC6A4 methylation was not associated with exposure to antidepressant drugs nor with depressive symptoms at Time 2. Newborns’ SLC6A4 methylation was lower at CpG sites 6 and 9 in association with higher levels of maternal depressed mood assessed at Time 1. Notably, similarly to maternal SLC6A4 methylation, the methylation status of the SLC6A4 promoter region in newborns was unaffected by maternal mood at Time 2 or exposure to antidepressant drugs. Maternal and newborn SLC6A4 methylation did not significantly correlate.
- Figure 3 about here -
The conjoint role of genetic (i.e., 5-HTTLPR) and environmental exposure to prenatal stress in SLC6A4 methylation was investigated in a study by Wankerl and colleagues (2014). Methylation was assessed at 83 CpG sites within the promoter region of the SLC6A4 gene in a cohort of human adults. Prenatal stress was retrospectively measured through maternal reports from a subset of participants’ mothers. The measured SLC6A4 mRNA expression was lower in individuals with an S genotype for 5-HTTLPR. Individuals exposed to maternal prenatal stress had significantly higher methylation at one CpG site out of 83 and lower SLC6A4 mRNA expression. The association between prenatal stress and mRNA expression was dose-dependent, on the basis of the number of stressful life events. Notably, no significant effect of prenatal stress and the 5-HTTLPR genotype was documented on the 15
mean SLC6A4 methylation. Moreover, no significant correlation emerged between SLC6A4 methylation and mRNA expression. Nonetheless, in one CpG site (i.e., CpG 9), a significant association emerged between methylation and expression of SLC6A4. 8.2.
Pain-related stress exposure Postnatal exposure to skin-breaking procedures during hospitalization in the neonatal
intensive care unit (NICU) was shown to associate with alterations of SLC6A4 methylation measured both soon after birth and during school age in children born very preterm (Chau et al., 2014; Montirosso et al., 2016; Provenzi et al., 2015). When compared at birth, SLC6A4 methylation, measured at 20 CpG sites adjacent to exon 1 (Nikolova et al., 2014), was not significantly different between very preterm infants and full-term infants (Provenzi et al., 2015). Nonetheless, methylation increased from birth to discharge at specific CpG sites as a function of the number of skin-breaking procedures to which preterm infants were exposed during their hospitalization. Notably, this effect was not affected by the length of NICU stay and perinatal variables. Using a micro-longitudinal approach, the above-mentioned altered methylation at the level of one specific CpG site (i.e., CpG5, Chr17: 28562847-28562848) was associated with a difficult temperamental style in very preterm infants at 3 months but not in their full-term counterparts (Montirosso et al., 2016). Chau and colleagues (2014) provided data on 7-year-old very preterm children, measuring SLC6A4 methylation within the promoter region (Devlin et al., 2010) and emotional problems through the Child Behavior Check List (CBCL). In children born very preterm, compared to their full-term counterpart, four CpG sites had significantly higher methylation levels (Chau et al., 2014). In order to reduce the number of comparisons, a principal component analysis (PCA) was carried out, leading to a principal component (PC1) which summarized the majority of CpG sites. PC1 accounted for 32% of the variance in SLC6A4 16
methylation and was significantly associated with VPT children’s total problems, which were not documented in full-term children. 8.3.
Childhood trauma
8.3.1. SLC6A4 methylation correlates of childhood traumas Beach and colleagues (2010) first reported the association between exposure to childhood abuse and altered methylation of the SLC6A4 gene. They focused on a 71-CpGsite island surrounding exon 1, but the main associations regarded a subset of sites from 1 to 17. Childhood abuse was measured retrospectively through self-reports in adulthood, covering both physical and sexual abuse (presence of any abuse vs. no abuse). Child abuse was associated with a pattern of hyper-methylation of the 1-to-17 CpG site component. In females, two specific CpG sites (i.e., CpG1 and CpG3) were significantly hyper-methylated in association with child abuse. In another study, the number of traumatic events was retrospectively obtained, including a wide range of adverse exposures from abuse to maltreatment, from the sudden death of a close friend or relative to any kind of serious injury (Koenen et al., 2011). The authors also obtained a measure of lifetime depression and post-traumatic stress (PTSD) symptoms. SLC6A4 methylation was measured at two CpG sites, one within the first exon and upstream of the TSS and the other within the first intron and downstream of the TSS. Nonetheless, results were reported only for the second CpG site. SLC6A4 single-site methylation was significantly associated with the number of traumatic events and being female but not with depressive and PTSD symptoms. However, while at low methylation, the association between the number of traumatic events and PTSD symptoms was stronger. At high level of methylation, no association was documented, which was interpreted as a marker of resilience. 17
Kang and colleagues (2013) assessed SLC6A4 methylation at 7 CpG sites within exon 1 in a sample of patients with major depression. Childhood adversities (including parental loss, family financial hardship, and physical and sexual abuse) from birth to 16 years were examined. All childhood adversities significantly associated individually with higher overall SLC6A4 methylation. Moreover, parental loss was associated with higher CpG2 methylation and the other adversities with CpG7 methylation. Notably, no association emerged between SLC6A4 methylation and the outcomes of a 12-week treatment with antidepressants. 8.3.2. Adversity-related change in SLC6A4 methylation and gene expression In an attempt to examine the association between abuse-related alterations of SLC6A4 methylation and the actual effects on gene transcription, Vijayendran et al. (2012) examined different portions of SLC6A4 for associations with childhood abuse and mRNA expression. No expression was detected at exon 1C and 1B, despite the fact that expression was detected when accounting for 1A together with 1B, and for all splice variants. Two consecutive CpG sites upstream of the TSS predicted gene expression for exon 1A and 1B, whereas methylation of two CpG sites within the CpG island were associated with total SLC6A4 expression. Sexual abuse was shown to be linked with a higher methylation level in two of these CpG sites. 8.3.3. Trauma-related changes in SLC6A4 methylation and 5-HTTLPR polymorphism The association between sexual abuse and SLC6A4 hyper-methylation at exon 1 was replicated, in women only, in a second study from Beach and colleagues (2011). Moreover, altered SLC6A4 methylation was a mediator of the association between childhood sexual abuse and later symptoms of antisocial personality disorder. Moreover, Beach and collaborators also tested for the conjoint role of SLC6A4 methylation and genetic variation (i.e., the 5-HTTLPR polymorphism). The significant association between childhood sexual 18
abuse and antisocial symptoms was present only in women with at least one short (S) allele of the 5-HTTLPR, which is known to convey a sensible increase in stress susceptibility, compared to the long (L) version. Van der Knaap and collaborators (2015) assessed SLC6A4 methylation and retrospective number of traumatic adverse experiences in a large cohort of adolescents, accounting also for 5-HTTLPR genetic variability. The authors accounted for perinatal adversities (e.g., maternal problems during pregnancy or 3 months after delivery, preterm birth, maternal alcohol and smoking during pregnancy) as well as for childhood trauma (e.g., sexual and physical abuse) and discrete stressful events reported by parents during childhood (i.e., 0-to-11 years) and adolescence (12-to-15 years). Perinatal adversities and both childhood trauma and stressful events did not associate with methylation measured at 14 to 15 years. Adverse life events experienced during adolescence were associated with higher SLC6A4 methylation independently of childhood exposure. Despite the fact that methylation levels did not differ between 5-HTTLPR genotypes, homozygous individuals for the L allele had greater association between stressful life events and methylation increase in adolescence compared to S-carriers. 8.3.4. Trauma-related changes in SLC6A4 methylation and brain function A recent study reported the association among SLC6A4, 5-HTTLPR, mRNA expression and hippocampal volume in human adults exposed to early traumatic events (Booij et al., 2015). Greater childhood abuse correlated with higher overall region SLC6A4 methylation, which was further linked to smaller hippocampal volume. Physical abuse was the adverse event most strongly associated with DNA methylation increase. Individuals with the LL genotype for the 5-HTTLPR and a history of abuse had greater SLC6A4 methylation
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compared to S-carriers or to individuals without a history of abuse. No significant association emerged between SLC6A4 methylation and mRNA expression. In a recent fMRI study on the visual emotional attention-shifting task (Frodl et al., 2015), SLC6A4 methylation was compared in depressed patients and healthy controls. Methylation was quantified at 11 CpG sites, and individuals were asked about any childhood traumatic experience. The level of SLC6A4 methylation did not associate significantly with groups and with performance in the visual emotional attention-shifting task (i.e., time reaction and number of incorrect valence judgment). Overall, participants with higher SLC6A4 methylation had increased activation of their left anterior insular cortex and left frontal inferior operculum compared to individuals with lower SLC6A4 methylation. When shifting attention away from negative stimuli, an opposite pattern of reduced activation of the right posterior insula and Rolando operculum were observed in high-methylation individuals compared to their low-methylation counterparts. When shifting attention from neutral stimuli, highmethylation individuals had less activation of the superior temporal lobe, whereas lower activation of the pons was observed when shifting attention away from positive stimuli. 8.3.5. Trauma-related changes in SLC6A4 methylation and stress reactivity Alexander and colleagues (2014) assessed the functional consequences of SLC6A4 methylation on stress reactivity in adults, controlling for the exposure to childhood traumatic events. Site-specific and overall average methylation was measured, encompassing 83 CpG sites within the promoter region. Information about childhood or recent traumatic events was obtained retrospectively through self-reporting. Subjects were exposed to a lab procedure to test for social and cognitive stress reactivity, namely the Trier Social Stress Test (TSST), and salivary cortisol was obtained before and after the procedure. No differences in salivary cortisol were observed between individuals showing low or high mean SLC6A4 methylation 20
levels. Nonetheless, a significant interaction emerged between SLC6A4 methylation and 5HTTLPR genotype: only in individuals with low levels of methylation, the presence of at least one S allele was associated with higher cortisol levels. Consistently, higher methylation levels were interpreted as a resilience factor, preventing genetically related susceptibility to stress. In a subsequent work from the same authors (Wankerl et al., 2014), childhood traumatic events, though not recently experienced ones, were associated with reduced SLC6A4 mRNA expression. Nonetheless, this association was not explained by the amount of SLC6A4 methylation measured as an overall mean level and at site-specific level. The TSST was used to test for stress reactivity in association with life events and DNA methylation of the SLC6A4 gene (Duman and Canli, 2015). The number of childhood traumatic events was obtained, including physical, sexual, and emotional abuse as well as physical and emotional neglect. Moreover, chronic stress during the last 3 months was also measured through the Trier Inventory of Chronic Stress (TICS), a self-report measure of the frequency of chronic stress behaviors. Salivary cortisol samples were obtained before and after the TSST procedure. The 5-HTTLPR genotype and the post-TSST SLC6A4 mRNA expression were obtained. SLC6A4 methylation was measured in 79 CpG sites. SLC6A4 methylation did not differ between different 5-HTTLPR genotypes. No associations emerged among SLC6A4 expression, methylation and childhood trauma. Nonetheless, methylation in a subset of CpG sites was related to early childhood traumatic exposures in S-carrier subjects but not in LL participants. Post-TSST mRNA expression of SLC6A4 was increased in LL individuals compared to S-carrier subjects, for whom it remained unchanged. Moreover, SLC6A4 expression was inversely correlated with the number of childhood traumatic events in S-carriers but not in LL individuals. As for stress reactivity, only in S-carrier individuals, higher SLC6A4 methylation predicted a higher cortisol response to the TSST. Finally, S21
carriers showed a significant association between SLC6A4 methylation in a subset of CpG sites and chronic stress. 8.4.
Peer-related trauma Only one study assessed the association between peer-related traumatic exposure
and methylation of the SLC6A4 gene, focusing on bullying victimization in childhood (OuelletMorin et al., 2013). Monozygotic twins were asked about bullying victimization, and SLC6A4 methylation was assessed at 44 CpG sites within the promoter region. Bullying victimization was assessed prospectively when the children were 7, 10, and 12 years old. SLC6A4 methylation was measured at 5 and 10 years. The TSST was used at 12 years, and salivary cortisol was obtained before and after the stressful procedure. Bullied twins had a blunted cortisol reaction to stressful exposure and higher site-specific (i.e., CpG 8) and overall average SLC6A4 methylation compared to non-bullied twins. Notably, the prospective nature of the study accounted for methylation before bullying victimization reports, showing that levels of SLC6A4 methylation at that time did not differ between twins. A site-specific (i.e., CpG 8) increase in methylation from age 5 to 10 years was observed only in bullied twins and was significantly correlated with the blunted cortisol response to the stressful task. 8.5.
Environmental stress Environmental stress is meant to encompass sources of adverse exposures not related
to discrete events, but rather to continuous exposure to adverse life conditions, such as socioeconomic disadvantages and work-related distress (Evans and Cohen, 1987). Recently, Beach and colleagues (2014) examined the relationship between cumulative exposure to low socio-economic status (SES) and SLC6A4 methylation. The exposure to low SES condition was assessed during pre-adolescence using primary caregiver reports and included a rate for family poverty, single-parent family, primary caregiver educational level and unemployment, 22
family receipt of temporary assistance for needy families, and income rated as not adequate to meet family needs. Families were characterized as having a low or high SES risk, mainly represented by family poverty. The 5-HTTLPR genotype was obtained, and methylation was measured in 16 CpG residues. No effects of genotype on methylation were observed. Only in females, one CpG site showed an increase of methylation in association with cumulative low SES risk. Methylation of five CpG sites within the SLC6A4 promoter region was measured in association with work-related stress in a cohort of 49 nurses (Alasaari et al., 2012). Workrelated stress, signs of burnout, and depression were measured through self-report instruments. The 5-HTTLPR genotype was also obtained. Nurses working in high-stress environments had significantly lower methylation levels at the investigated five CpG sites compared to nurses from low-stress environments. No effects of the 5-HTTLPR genotype was documented. Burnout and work-related stress were significantly associated with the change in methylation levels. 9. DISCUSSION In the present review, the existing literature on the association between SLC6A4 methylation and adverse exposures in humans was systematically analyzed. Nineteen papers were included. On the one hand, the results corroborate findings from research on animals about the potential role of SLC6A4 epigenetic modifications as a means through which the exposure to adverse environments might be embedded into an individual’s biology, further programming socio-emotional functioning and stress susceptibility. On the other hand, a wide heterogeneity was documented regarding the adopted methylation techniques and analysis, as well as for sample characteristics and the adverse events that were investigated. In the following paragraphs, we sum up the evidence for the association of adverse exposure with 23
altered SLC6A4 methylation and for the relationship between changes in SLC6A4 methylation and further phenotypic outcomes. Moreover, open questions and critiques about SLC6A4 human epigenetic research are addressed. 9.1.
Is the exposure to adverse events associated with altered SLC6A4 methylation? As mentioned, there is a growing body of studies suggesting that exposure to early
traumas in childhood (e.g., physical and sexual abuse, as well as parental loss and other adverse events) results in a pattern of hyper-methylation at specific CpG sites located within the SLC6A4 exon 1 (Beach et al., 2010; Kang et al., 2013; Vijayendran et al., 2012; Koenen et al., 2011). As such, a pattern of increased SLC6A4 methylation appears to be promising as a potential marker of adversity exposure in humans, despite some results showing sitespecific decrease in SLC6A4 methylation (Alasaari et al., 2013; Van der Knaap et al., 2015). As such, the possibility that exposure to adversities might result in either an increase or a decrease in serotonin transporter gene methylation could not be ruled out and still warrants further investigation. As suggested by Booij et al. (2013), differences in methylation changes might be related to the type of adversity as well as to individual characteristics, including sex and age (Beach et al., 2010; Booij et al., 2015). At first, conflicting results might be an effect of different measures of adversities (Van der Knaap et al., 2015), as well as a function of stressor type, duration and timing (Booij et al., 2013). More generally, the presence of discordant “directions of change” in SLC6A4 methylation following exposure to adverse events should be considered as a biologically-based warning against reductionist interpretation of DNA methylation as a direct marker of detrimental developmental conditions (Provenzi and Montirosso, 2015; Richardson et al., 2014). Unfortunately, to date, it is not possible to determine the functional effects of SLC6A4 methylation changes and whether an increase or a decrease in methylation levels has univocal implications for childhood and 24
adulthood health and disease. A tentative interpretation is that an adversity-related increase in SLC6A4 methylation might be indicative of a biomarker for that event, suggesting that the encounter with an adverse environmental condition was registered by the body, whereas a decrease would suggest a more direct movement toward a biologically protective state with regard to the risk of developing negative outcomes later in life (Alasaari et al., 2012). Nonetheless, assuming a more conservative view, it still remains difficult to firmly conclude whether hyper- and hypo-methylation reflect our attempt to adapt to the environment or constitute a risk factor for diseases later in life (Alexander et al., 2014). 9.2.
Does altered SLC6A4 methylation associate with adverse outcomes? As for the outcomes of altered SLC6A4 methylation for socio-emotional functioning and
stress regulation, studies are only partially consistent. First, 8 out of 16 papers reported developmental outcomes in associations with adversity-related SLC6A4 methylation status. Higher methylation was found to be related to higher global behavioral problems at school age in children (Chau et al., 2014), with reduced hippocampal volume in adults (Booij et al., 2015) and with a peculiar pattern of left and right cerebral activations in response to emotionally relevant stimulations (Frodl et al., 2015). Nonetheless, Duman and Canli (2015) did not document any association between the status of SLC6A4 methylation after exposure to early traumatic events and the cortisol response to lab-elicited cognitive and emotional stressful conditions. In another study, this association has been found to be mediated by genetic stress susceptibility conveyed by 5-HTTLPR (Alexander et al., 2014). Individuals with a low level of methylation were found to produce higher cortisol concentrations in response to the same lab procedure, but only if they were carrying at least one S allele of the 5-HTTLPR polymorphism. Notably, having a high level of methylation of the SLC6A4 gene emerged as a resiliency-conveying factor. This was also suggested by the single CpG study by Koenen and 25
collaborators (2011). Here, individuals with low levels of methylation at a single CpG site located in the first intronic region of SLC6A4 showed a significant association between early stressful events and a greater risk of post-traumatic stress disorder, which was not documented in individuals with high levels of methylation. As for the risk of developing psychopathological outcomes, Kang and colleagues (Kang et al., 2013) did not document any significant association with the outcome of depression, whereas Beach and colleagues (2011) found a positive association between SLC6A4 methylation and symptoms of antisocial personality disorders, but this was ultimately true only for S-carrier women. We suggest that a meta-analytic study of the effects of adversities on SLC6A4 methylation status should be pursued as a next step in order to advance our confidence in SLC6A4 epigenetic change (increase/decrease) as a reliable marker of early adversity exposures. In sum, despite the fact that increased methylation has been shown to be a potential marker of adversity exposure, conclusive take-home messages about the role of SLC6A4 adversity-related changes in methylation seem to be difficult to draw in a univocal way. The pathways through which adversities might result in altered serotonin transporter gene methylation and further adverse developmental outcomes need additional investigations. Furthermore, several methodological and study design limitations can affect findings, leaving several critical issues unsolved. 10. OPEN QUESTIONS 10.1. Open question #1: Issues with DNA methylation measurement Methylation patterns are, to a large extent, tissue-specific (Wang et al., 2012). This means that it could not be taken for granted that differences in SLC6A4 methylation observed in peripheral tissues in humans are indicative of serotonin availability in the central nervous system. Indeed, an important caveat with human behavioral epigenetic studies is that 26
researchers have no access to brain tissues in living individuals. As such, DNA methylation patterns need to be assessed indirectly on peripheral tissues, including blood, saliva, and epithelial cells. Finally, it should be considered that in some studies methylation was obtained from whole blood samples rather than specific cells (e.g., leukocytes). If this is thought to reduce the risk of altering DNA methylation conveyed by different cell-specific techniques, it should be considered that the presence of a heterogeneous mix of cells might be viewed as a potential confounder. Nonetheless, as reported by Booij and colleagues (2013), researchers should not be dissuaded. Genome-wide studies have revealed that DNA methylation levels between blood and post-mortem brain tissues are likely to be highly correlated (Horvath et al., 2012). Moreover, some stress-related genes have shown to be less tissue-specific than others, and SLC6A4 appears to be among these genes. For example, significant correlations have been found between SLC6A4 kinetics in different cells from blood, nerve endings, and brain synaptosomes, suggesting that peripheral and central samples might be associated (Rausch et al., 2005; Uebelhack et al., 2006). Moreover, indirect evidence has come from neuroimaging studies. Frodl and colleagues (2015) showed that methylation of SLC6A4 from peripheral blood was linked with anatomic features in brain, supporting the hypothesis that peripherally-observed changes in DNA methylation may provide functionally relevant information about brain regions involved in socio-emotional functioning. Similar confirmations were recently provided for saliva samples (Thompson et al., 2013). As such, to the best of our knowledge, peripheral tissues appear to be the closest reliable source of functionally relevant DNA methylation information in relation to exposures to adverse events in humans. Furthermore, it is worth noting that the association between SLC6A4 methylation in peripheral tissues and the actual expression of 5-HTT has received inconsistent evidence. 27
For example, Wang and colleagues (2012) documented that SLC6A4 methylation in peripheral tissues was linked to 5-HTT expression. However, Duman and Canli (2015) did not find evidence that methylation in a subset of CpG sites within the exon1 CpG island correlated significantly with mRNA expression, which confirmed previous reports (Wankerl et al., 2014). To date, the actual relationship between SLC6A4 methylation and mRNA expression appears to be rather complex, and it might be affected by genetic variations at the level of the upstream 5-HTTLPR polymorphism (Philibert et al., 2007). Nonetheless, some studies failed to prove a genetically related effect of SLC6A4 methylation on mRNA expression (Wankerl et al., 2014), and this non-replication might be caused, at least partially, by faster degradation of mRNA markers compared to DNA methylation patterns (Avila et al., 2010). The latter would be considered as indirect evidence that methylation at peripheral cells might be a more reliable biomarker for stress exposures compared to mRNA (Bird, 2002). The majority of the studies focused on the promoter region of SLC6A4, widely overlapping with the exon 1 CpG island. Nonetheless, different portions of this region have been targeted by different researchers. The choice of different regions should be carefully addressed by researchers (Booij et al., 2015), since considering different segments of SLC6A4 promoter region might result in different links with developmental outcomes, based on the different binding attractiveness of specific CpG sites (Sugawara et al., 2013). Moreover, it has been recently suggested that intronic regions might also have a role in regulating gene expression (Lomelin et al., 2010), and this should warrant renewed attention in further studies. 10.2. Open question #2: Polymorphism-related differences in SLC6A4 methylation It has been suggested that early life exposure might be linked to site-specific SLC6A4 methylation as a function of genetic variation (i.e., the 5-HTTLPR polymorphism). 28
Theoretically, it might be hypothesized that the S allele, conveying higher susceptibility to stress by reducing the rate of inter-synaptic serotonergic turnover (Canli and Lesch, 2007) and heightened SLC6A4 methylation, resulting in reduced availability of 5-HTT (Lesch, 2011), would have an additive effect on serotonin transporter expression. The issue of whether SLC6A4 methylation and 5-HTTLPR genetic variations significantly interact has received attention in recent literature, but we are still far from a convincing unifying comprehension of this interplay. While Booij and colleagues (2015) and Beach and collaborators (2014) did not document any significant association between the SLC6A4 genotype and DNA methylation, Alasaari et al (2012) showed higher methylation in S-carriers. Others have provided evidence for both an adversity-to-methylation relationship, mediated by 5-HTTLPR variability, as well as a polymorphism-to-methylation link, conditioned by the SLC6A4 methylation level. For example, L-homozygous individuals had stronger association between stress exposure measured in adolescence and methylation increase at 14 to 15 years (van der Knaap et al., 2015). Exposure to early life stressors correlated positively with methylation at SLC6A4 but only in S-carrier individuals (Duman and Canli, 2015). Moreover, L-homozygotes had lower methylation than S-carriers but only at low levels of stress exposure (van der Knaap et al., 2015). Again, S-carrier women with exposure to high socio-economic risk had higher methylation compared to S-carriers with low socio-economic risk and with their L-homozygous counterparts (Beach et al., 2014). Finally, low methylation was related to increased cortisol reactivity to stress in S-carrier individuals, but this association was not documented in the presence of high methylation (Alexander et al., 2014). In sum, allelic-specific methylation should be a mechanism through which a stressful environment can lead to vulnerability (van der Knaap et al., 2015), but more research is needed to gain understanding of the complex
29
interplay between genetically determined and epigenetically acquired variations at the serotonin transporter gene. 10.3. Open question #3: Sample characteristics and SLC6A4 methylation Features of participants might account for variability among different studies. First, the sample size widely varied among papers included in the present review, which suggests very different levels of statistical power (Duman and Canli, 2015). Moreover, the age at which methylation is measured should not be considered secondary. A trend for methylation increase with aging has been recently suggested to be gene-specific (Jung and Pfeifer, 2015). Moreover, alterations in the availability of central nervous system serotonin may result in very distinct impacts on the organism during different developmental time windows (Homberg et al., 2010). As such, it could be suggested that the timing of methylation assessment should be decided with strictness and should be consistent with a well-grounded rationale (Alexander et al., 2015). Another issue regards the hypothesis of sex-related differences in the methylation status of the serotonin transporter gene. Again, inconsistent findings arise from the studies included in the present review. Some researchers reported higher methylation (Van der Knaap et al., 2015) and more robust associations between adverse events exposure and SLC6A4 increase (Beach et al., 2014) in women, whereas others reported higher methylation in men (Booij et al., 2015). Nonetheless, these controversies seem to suggest that there is no convincing evidence that sex-related differences should have a critical role in regulating SLC6A4 gene methylation in humans (Booij et al., 2015). 10.4. Open question #4: Limitations of study design The study design is another important issue for interpretation of the SLC6A4 methylation findings related to adverse exposure and socio-emotional outcomes in humans. 30
Studies included in this review were in part cross-sectional, whereas the greatest part adopted a retrospective design, measuring methylation at the time of outcome assessment and
collecting
self-reported
information
about
previous
exposures
to
adversities.
Retrospective investigation provides a potential temporal direction for the interpretation of significant associations. Nonetheless, this kind of study design is correlational in its nature, which does not allow for a causal interpretation (Duman and Canli, 2015). Therefore, this methodological issue might, at least partially, account for some inconsistencies between studies showing methylation changes in relation to previous childhood traumas (Beach et al., 2010) and others failing to replicate these findings (Van der Knaap et al., 2015). A call-foraction for longitudinal prospective studies is strongly recommended by all the papers in this review (see, for example, Frodl et al., 2015). From this point of view, the application of epigenetic investigation to the field of preterm birth (Preterm Behavioral Epigenetics [PBE]; Montirosso and Provenzi, 2015) appears to be promising. In fact, it can be a priori hypothesized that preterm infants will be hospitalized in a NICU and therefore exposed to variable amount of physical, painful and socio-emotional stress due to the nature of the NICU environment. As such, longitudinal prospective studies are encouraged in this area and it is warranted that future PBE research include an investigation of the epigenetic vestiges of both stressful and protective (i.e., developmental care practices) factors of NICU stay. 11. CONCLUSIONS SLC6A4 methylation appears to be a widely reported biomarker for exposure to adversities early in life as well as during adolescence and adulthood. Nonetheless, in order to develop a full understanding of the pathways through which epigenetic mechanisms occur at the level of the serotonin transporter gene, future research needs to address specific issues. Keeping in mind the aforementioned open questions, at the actual state of the art the main 31
critical points regard (1) improvements at the level of methylation assessment, (2) study design and methodological caveats, and (3) specific investigations of genome–epigenome interplay. Broadly speaking, the rapid spreading of human behavioral epigenetic studies holds the risk of producing a growing number of findings, which could hardly be merged together in a coherent theoretical framework. As such, we recommend that researchers engaged in the study of SLC6A4 epigenetic markers in association with specific adversity conditions develop a prospective model grounded in previous literature to guide future research projects and uncover new areas of investigation. A clinically relevant and theoretically grounded approach to human behavioral epigenetics is warranted to enhance the research resource-to-outcomes balance, limiting the risk of favoring misleading interpretations of reductive epigenetic results (Richardson et al., 2014).
32
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39
Figure 1. Structure of the SLC6A4 gene.
40
Figure 2. Selection criteria flow chart.
Note. Duplicates were determined in two different ways: (a) Duplicates within the same database, such as a double entry for a single paper in Scopus; (b) Duplicates between two or three different databases. Out-of-focus records included papers being conducted on animals (N = 6) or papers on humans which did not assessed SLC6A4 in association with exposure to any adverse event (N = 45). These latter included papers reporting on: SLC6A4 methylation in association with disease conditions, N = 28; SLC6A4 associations with drug treatments, N = 6; methylation techniques, N = 4; SLC6A4 polymorphic variations, N = 3; genome-wide DNA methylation, N = 1). Out-of-focus records included also 3 papers which reported data already published in other papers which were included in the present review.
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Figure 3. Graphic representation of SLC6A4 gene portions targeted by the studies included in the review. The squared portion highlights sites surrounding the CpG island within exon 1.
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Table 1. Studies included in the review.
Studies st
Year
1 author
2010
Beach
2010
Devlin
2011
Beach
2011
Koenen
2012
Alasaari
2012
Vijayendran
2012
Wang
2013
Kang
2013
OuelletMorin
2014
Alexander
2014
Beach
2014
Chau
2014
Wankerl
2015
Booij
2015
Duman
SLC6A4 methylation information Tissue Peripheral blood Peripheral/cord blood Peripheral blood Whole blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Buccal cells
Description of region
CpG sites N
Unit of analysis
Surrounding Exon 1
71
Adjacent to Exon 1A
Adverse event information
N
Age at investigation
Type
Age at adverse event
PCA + CpG sites
192
Adults
Abuse
Childhood
10
CpG sites
82
Newborns
Maternal depression
Prenatal
Surrounding Exon 1
71
Average
155
Adults
Abuse
Childhood
Exon 1 and Intron 1
2
CpG sites
100
Adults
Childhood
Promoter region
5
PCA
49
Adults
Trauma Environmental stress
Post-TSS, Exon 1, 5'UTR, 3'UTR
14
CpG sites
152
Adults
Abuse
Childhood
Upstream of TSS
24
CpG sites
25
Adults
Aggression
Childhood
Promoter region
7
102
Adults
Traumatic events
Childhood
Promoter region
12
44
Children
Bullying
Childhood
186
Adults
388
Adults
111
Children
85
Adults
69
Adults
71
Adults
Peripheral blood Peripheral blood
Promoter region
83
Post-TSS, Exon 1, 5'UTR, 3'UTR
16
Saliva
Adjacent to Exon 1A
10
Promoter region
83
Promoter region
24
Upstream to TSS
79
Peripheral blood Peripheral blood Peripheral
Sample information
CpG sites + Average CpG sites + Average CpG sites + Average CpG sites CpG sites + PCA + Average CpG sites + Average CpG sites + Average FA + Average
43
Traumatic events Environmental stress Pain Traumatic events Traumatic events Traumatic
Adulthood
Childhood Childhood Infancy Childhood Childhood Childhood
blood
events
2015
Frodl
Whole blood
Promoter region
11
CpG sites + Average
60
Adults
Maltreatment
Childhood
2015
Provenzi
Peripheral/cord blood
Adjacent to Exon 1A
20
CpG sites
88
Newborns + Infants
Pain
Perinatal
2015
van der Knaap
Whole blood
Exon 1
11 (units)
CpG sites + Average
939
Adolescents
Traumatic events
Adolescence
Peripheral/cord Newborns + Adjacent to Exon 1A 20 CpG sites 78 Pain Perinatal blood Infants Note. CpG sites = DNA sites rich in cytosine and guanine; CpG units = groups of adjacent CpG sites; PCA = Principal Component Analysis; FA = Factor Analysis. 2016
Montirosso
44
