New-born females show higher stress- and genotype-independent methylation of SLC6A4 than males
© Dukal et al.; licensee BioMed Central. 2015
Received: 5 August 2014
Accepted: 24 March 2015
Published: 15 April 2015
Research has demonstrated an association between exposure to early life stress and an increased risk of psychiatric disorders in later life, in particular depression. However, the mechanism through which early life stress contributes to disease development remains unclear. Previous studies have reported an association between early life stress and altered methylation of the serotonin transporter gene (SLC6A4), a key candidate gene for several psychiatric disorders. These differences in methylation are influenced by sex and genetic variation in the SLC6A4-linked polymorphic region (5-HTTLPR). Furthermore, one study indicated that stress during pregnancy may induce methylation changes in SLC6A4 in the newborn. The present study is the first to investigate whether early life stress during pregnancy impacts on SLC6A4 methylation in newborns, taking into account the influence of genetic variation and sex.
Cord blood was obtained from newborns with high (n = 45) or low (n = 45) early life stress, defined as maternal stress during pregnancy. The effect on methylation of early life stress, 5-HTTLPR genotype, and sex was assessed at four cytosin-phosphate-guanine dinucleotide (CpG) sites in the promoter associated CpG island north shore (CpG 1 to 4). The epigenetic analyses focused on these CpG sites, since research has shown that CpG island shore methylation has functional consequences.
Significant sex-specific methylation was observed, with females displaying higher methylation levels than males (p < 0.001). Importantly, this effect was influenced by neither early life stress nor genotype.
The present data suggest that sex-specific methylation of SLC6A4 is present at birth, and is independent of early life stress and 5-HTTLPR genotype. This may contribute to the sex-specific prevalence of depression.
KeywordsSerotonin transporter SLC6A4 5-HTTLPR Methylation Early life stress Sex
The serotonin transporter gene (SLC6A4) is one of the most extensively investigated genes in psychiatry, and has been implicated in a wide range of psychiatric disorders . Research has shown that variation in this gene is associated with stress-related psychiatric disorders, such as major depression and anxiety . The most consistently reported association is with variation in the SLC6A4-linked polymorphic region (5-HTTLPR). This 5-HTTLPR consists of a 44-base-pair insertion/deletion polymorphism in the 5′ regulatory promoter region of SLC6A4, which results in a short (S) and long (L) allele of the gene and a single nucleotide polymorphism in the L allele of 5-HTTLPR (rs25531 A/G) which leads to a triallelic locus (S/LA/LG) . The S allele has been reported to reduce transcriptional activity of the gene promoter in vitro, and to be associated with depression-related personality traits [4,5]. Moreover, research has identified a gene x environment interaction for life stress and the 5-HTTLPR genotype in adults with depression . In comparison to L homozygotes, S allele carriers showed higher levels of depressive symptoms, diagnosable depression, and suicidality.
Furthermore, numerous studies have demonstrated methylation differences in SLC6A4 in individuals with depressive symptoms , depression , and a history of early life stress [9-14]. Sex- [8,9] and genotype-effects have also been described [10-12].
Several studies have demonstrated that early life stress has a major impact on mental health in later life. Childhood adversity is associated with increased vulnerability to several psychiatric disorders, in particular mood, anxiety, and personality disorders . Furthermore, maternal stress during pregnancy has been associated with distinct behavioral disturbances in the offspring in later life . In particular, maternal stress during pregnancy appears to have major effects on the stress response and the propensity to depression in the offspring . To date, only one study has analyzed the influence of depressed maternal mood during pregnancy on serotonin transporter methylation levels in the newborn. In this study, increased 2nd trimester depressed maternal mood was associated with decreased maternal and infant serotonin transporter promoter methylation . However, the authors did not take into account the possible effect of variation in 5-HTTLPR genotype and sex.
A previous study has shown that methylation of SLC6A4 CpG island shore CpG sites has functional consequences . Generally, methylation of DNA cytosines in the context of cytosin-phosphate-guanine (CpG) dinucleotides is a covalent modification of DNA with a methyl group. The latter is introduced into the DNA by an enzymatic reaction catalyzed by DNA methyltransferases . CpG dinucleotides are often clustered in CpG islands at the promoter region of genes. In general, methylation leads to gene silencing via reduced trancriptional activity . Previous authors have hypothesized that early life adversity results in DNA methylation and life-long phenotypes .
Therefore, the aims of the present study were to determine 1) whether exposure to maternal stress during pregnancy leads to higher methylation levels in SLC6A4 in newborns; and 2) whether this is influenced by sex and 5-HTTLPR genotype. The analyses focused on four previously reported functional serotonin transporter gene CpG island north shore CpG sites .
Data were obtained from a cohort of mothers and their infants (n=410), respectively recruited during the third trimester of pregnancy (4–8 weeks prior to term) from the Rhine-Neckar Region of Germany. The study protocol was approved by the Ethics Committee of the Medical Faculty Mannheim of the University of Heidelberg. The study was conducted in accordance with the Declaration of Helsinki. All mothers provided written informed consent prior to participation. A structured interview and questionnaires were used for risk factor assessment. Inclusion criteria for mothers were: main caregiver; German-speaking; and age 16–40 years. Exclusion criteria for mothers were: hepatitis B, hepatitis C, or human immunodeficiency virus (HIV) infection; any current psychiatric disorder requiring inpatient treatment; any history or current diagnosis of schizophrenia/psychotic disorder; or any substance dependency other than nicotine during pregnancy. Exclusion criteria for infants were: birth weight < 1,500 gram; gestational age < 32 weeks; or the presence of any congenital diseases, malformations, deformations, and/or chromosomal abnormalities.
Phenotypic assessment of mothers and infants at two time points
Prenatal/3 rd trimester
Exposure to ELS
Perceived stress (PSS) 
Pre- and perinatal complications
Prenatal distress (PDQ) 
Perinatal stressors (e.g., asphyxia, cesarian, preterm birth)
Life events (LES) 
Pregnancy & obstetric history (birth weight, gestational age, birth complications)
Social support (Soz-U.) 
Maternal health risk behavior (e.g. smoking)
Maternal mental & physical health
Maternity log-book data
Semi-standardized neuropsychiatric diagnostic interview (MINI) 
Depression screening (EPDS) 
Individual & family history of metabolic and other medical disorders
Psychopathology and socioeconomic-, psychosocial-, and perceived stress of the extreme group mothers (all data: mean ± SD or percentage)
High prenatal ELS (n = 45)
Low prenatal ELS (n = 45)
12.33 ± 4.70
1.27 ± 1.45
51.72 ± 8.05
27.41 ± 3.87
47.93 ± 8.41
28.04 ± 3.58
4.11 ± 2.14
0.53 ± 0.72
Current psychiatric disorder2
30.84 ± 5.64
12.84 ± 4.1
19.95 ± 7.56
5.95 ± 2.95
Socioeconomic and psychosocial stress
LES-negative events Score1
6.75 ± 4.39
1.80 ± 1.29
39.58 ± 3.85
51.89 ± 4.32
Living without a partner2
Separation(s) in the last year2
Physical conflicts within the preceding 12 months2
Composition of household > one person/room2
No academic qualification2
No professional education2
Monthly income per household ≤ 1,750 Euro2
To obtain a homogeneous composite measure of prenatal stress, a principal component analysis (PCA) was performed using the eight main stressor variables and the total adversity score. This yielded a first principal component (PC1) that explained around 60% of the common variance. PC1 was then used to determine two extreme groups: 45 infants (24 females; 21 males) with extremely high levels of prenatal early life stress; and 45 infants (27 females; 18 males) with extremely low levels of prenatal early life stress .
Demographic characteristics included in the methylation analysis were maternal age (maternal age high prenatal stress, 29.60 ± 5.61; maternal age low prenatal stress, 32.73 ± 3.69) and the sex of the newborn (high prenatal stress female 53.3%; low prenatal stress female 60%).
5-HTTLPR rs25531 multimarker genotyping
Simultaneous genotyping of 5-HTTLPR and rs25531 was carried out by polymerase chain reaction (PCR) according to Wendland et al. . In a total volume of 20 μl, 60 ng of genomic DNA was amplified in the presence of 1 x Master Mix (Promega) using the following primers: forward primer: 5′-TCCTCCGCTTTGGCGCCTCTTCC-3′; reverse primer: 5′-TGGGGGTTGCAGGGGAGATCCTG-3′. The PCR conditions were: 5 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 90 seconds at 70°C, 60 seconds at 72°C, and a final extension of 10 minutes at 72°C. Subsequently, complete PCR product was digested with HpaII (New Enlgand Biolabs) in a 25 μl reaction assay containing 1xNEBuffer 1 at 37°C for 4 h. Then, 15 μl of the digested PCR products were run on a 2.5% agarose gel and visualized with ethidium bromide staining. For the 5-HTTLRP L allele, a band of 512 basepairs (rs25531, A allele, uncut) or 402 basepairs and 110 basepairs (rs25531, G allele, cut) was visible. For the 5-HTTLRP S allele, a band of 469 basepairs was visible. The 5-HTTLPR rs25531 multimarker genotype is combined according to its SLC6A4 mRNA expression into high expressing genotype 5-HTTLPR LA and low expressing genotype 5-HTTLPR S and LG.
Two-tailed t-tests for independent samples (SPSS® Statistics 20) were used to compare demographic factors between the extreme groups. Nominal significance was set at α = 0.05. Data and results are expressed as means ± standard deviation (SD) or as percentages, as appropriate . Quality control and association testing of methylation data were performed using the R 2.15.3 software package (http://www.r-project.org). Association tests were performed using multifactorial ANOVA models which included all variables of interest with correction for plate effects. Tests were performed for each of the four sites and for the mean of all four sites.
P-values and r-squared values (in parentheses) obtained from multi factorial ANOVA models of the effect of sex, ELS, and 5-HTTLPR genotype on SLC6A4 methylation
Predictor in model
Mean across sites
Sex x ELS
Sex x Genotype
ELS x Genotype
P-values and r-squared values (in parentheses) obtained from multi factorial ANOVA models of the effect of sex, ELS, and combined 5-HTTLPR rs25531 genotype grouped according to Wendland et al. [ 23 ] on SLC6A4 methylation
Predictor in model
Mean across sites
Sex x ELS
Sex x Genotype_HL
ELS x Genotype_HL
The aims of the present study were to determine whether exposure to early life stress – defined as maternal stress during pregnancy – impacts SLC6A4 methylation levels in newborns, and whether this is influenced by sex or 5-HTTLPR genotype. In the present cohort, methylation was influenced by sex, but not by early life stress or 5-HTTLPR genotype. Following the integration of the effects of rs25531, which has been reported to influence expression of SLC6A4 , the results were unaltered.
Research has shown that DNA methylation can lead to persistent alterations in gene function, and that this may result in a range of psychiatric phenotypes . Robust data suggest that stress has an influence on methylation, and higher SLC6A4 methylation has been associated with reduced gene expression . In individuals with the S/S genotype of 5-HTTLPR, Sugawara et al. found an inverse correlation between SLC6A4 methylation levels and gene expression . Several earlier studies had demonstrated that early life stress-induced methylation differences were influenced by genetic variation in 5-HTTLPR genotype. In the Iowa adoptee sample, early adversity was associated with differential methylation, as mediated by 5-HTTLPR genotype [9-12].
As reported with high SLC6A4 methylation, the S allele of the 5-HTTLPR genotype leads to decreased gene expression, and has been repeatedly associated with an increased vulnerability for stress-related psychiatric disorders, such as depression and anxiety . However, the present study analyzed different CpG sites, and thus cross-study comparison of results is problematic. The present study focused on the functional CpG sites reported by Sugawara et al. . These were selected on the basis of their promising position in the island north shore and their reported functionality.
Sensitivity to stress changes throughout the life-span, and young adults with a history of stressful life events during the first five years of life show a particularly pronounced stress response . Therefore we expected individuals exposed to stress during an even earlier period of life to show increased methlyation compared to those with no history of exposure. Interestingly, however, in the present cohort, neither early life stress nor genotype influenced methylation in utero.
However, a strong sex-effect was apparent, and recent studies have generated independent evidence for an effect of sex on SLC6A4. Wang et al. reported that at birth, methylation of CpG site two was higher in females than in males . In a longitudinal assesssment of their cohort, the authors found persistent sex-differences two years after birth. In another study, Xu et al. found sex differences in this CpG site in the post-mortem adult brain . Neither of these studies, however, had taken into account early life stress or 5-HTTLPR genotype. Sex specific DNA methylation, i.e., other than that occurring during the X-inactivation process, has been demonstrated in a number of locus-specific () and genome-wide analyses (e.g. ). This could represent a mechanism underlying the differential susceptibility to psychiatric disorders observed between males and females . Further studies are warranted to determine both whether these methylation differences are permanent, and the contributory factors.
To our knowledge, the present study is the first to demonstrate sex-determined methylation of SLC6A4 in newborns, irrespective of early life stress status and 5-HTTLPR genotype. Given the previously reported functionality of SLC6A4 methylation and the importance of the serotonin transporter (5-HTT) in depression, higher methylation levels during early life may contribute to the increased risk for depression observed among females.
This work was supported by an Era-Net Neuron grant to Michael Deuschle, Marcella Rietschel and Manfred Laucht; and grant 01GS08147 (Marcella Rietschel) from the National Genome Research Network (NGFN-plus) of the German Federal Ministry of Education and Research (BMBF). Michael Deuschle, Marcella Rietschel and Manfred Laucht received support from the Dietmar-Hopp Foundation. We thank Christine Schmael for her critical reading of the manuscript. The authors thank the families and the midwives for their participation.
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