Behav Genet DOI 10.1007/s10519-015-9721-y

ORIGINAL RESEARCH

Alterations of Glucocorticoid Receptor Gene Methylation in Externalizing Disorders During Childhood and Adolescence Angela Heinrich1,2 • Arlette F. Buchmann3 • Katrin Zohsel3 • Helene Dukal1 • Josef Frank1 • Jens Treutlein1 • Vanessa Nieratschker1,4 • Stephanie H. Witt1 • Daniel Brandeis3,5 • Martin H. Schmidt3 • Gu¨nter Esser6 • Tobias Banaschewski3 Manfred Laucht3,6 • Marcella Rietschel1



Received: 14 July 2014 / Accepted: 7 April 2015 Ó Springer Science+Business Media New York 2015

Abstract Epigenetic modulations are a hypothesized link between environmental factors and the development of psychiatric disorders. Research has suggested that patients with depression or bipolar disorder exhibit higher methylation levels in the glucocorticoid receptor gene NR3C1. We aimed to investigate whether NR3C1 methylation changes are similarly associated with externalizing disorders such as aggressive behavior and conduct disorder. NR3C1 exon 1F methylation was analyzed in young adults with a lifetime diagnosis of an externalizing disorder

Edited by Carol Van Hulle. Angela Heinrich, Arlette F. Buchmann, Manfred Laucht, and Marcella Rietschel have contributed equally to this work. & Angela Heinrich [email protected] 1

Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

2

Department of Cognitive and Clinical Neuroscience, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Square J 5, 68159 Mannheim, Germany

3

Department of Child and Adolescent Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

4

Department of Psychiatry and Psychotherapy, University of Tuebingen, Tu¨bingen, Germany

5

Department of Child and Adolescent Psychiatry, University of Zu¨rich, Zurich, Switzerland

6

Department of Psychology, University of Potsdam, Potsdam, Germany

(N = 68) or a depressive disorder (N = 27) and healthy controls (N = 124) from the Mannheim Study of Children at Risk. The externalizing disorders group had significantly lower NR3C1 methylation levels than the lifetime depressive disorder group (p = 0.009) and healthy controls (p = 0.001) This report of lower methylation levels in NR3C1 in externalizing disorders may indicate a mechanism through which the differential development of externalizing disorders as opposed to depressive disorders might occur. Keywords Epigenetic  Glucocorticoid receptor  Methylation  Externalizing disorders  Adolescents

Introduction Research suggests that lower gene expression secondary to DNA methylation is an important link between environment and health (e.g. Weaver 2007). Since exposure to adversity is a reported methylation-mediating environmental factor, the major stress-response regulating biological system, i.e. the hypothalamic–pituitary–adrenal (HPA) axis, is of particular interest in this context (e.g. Booij et al. 2013; Holtzman et al. 2013). A key molecule of the HPA axis is the glucocorticoid receptor, which mediates the negative feedback response after binding to cortisol. Previous research has shown that exposure to early adversity leads to a persistent alteration of the glucocorticoid receptor in rodents and humans (e.g. Miller et al. 2009). This property renders the glucocorticoid receptor gene NR3C1 a key candidate in epigenetic investigations of psychiatrically relevant traits (e.g. Labonte et al. 2012; McGowan et al. 2009; Oberlander et al. 2008). The NR3C1 gene is characterized by high variability in the 50 -region. In

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humans, this region contains nine alternative non-translated first exons (1A–I) (e.g. Turner et al. 2010). In the case of exons 1B, 1C, 1D, 1F, and 1H, the genomic sequences that lie immediately upstream constitute promoters (Cao-Lei et al. 2011), which confer a tissue-specific expression pattern in the corresponding glucocorticoid receptor mRNA isoforms (Turner et al. 2008). Research has demonstrated that DNA methylation regulates the expression of the glucocorticoid receptor by altering the binding of transcription factors in the promoter regions (e.g. McGowan et al. 2008; Weaver et al. 2004). In a previous human study, a putative transcription factor binding site, which corresponds to a stress-responsive nerve growth factor inducible protein A (NGFI-A) binding site identified in the rat exon 17 promoter (Weaver 2007), was mapped to the human 1F promoter (McGowan et al. 2009). The results demonstrated that exon 1F was expressed in a tissuespecific manner in the hippocampus, as well as in plasmacytoid dendritic- and CD19? B cells (Turner and Muller 2005). Previous reports have shown that early adverse experiences increase the risk of mental disorder in later life (e.g. McEwen 2003; Mullen et al. 1996). Numerous studies have reported adverse mental health effects secondary to stress during intrauterine development (e.g. Walker et al. 2011), and this factor has been associated with schizophrenia (Weinstock 2001) and depression (Brown et al. 2000; Watson et al. 1999). Moreover, the impact of stress on mental health appears to persist throughout the life-span (Lupien et al. 2009). An important example of early life stress is adverse rearing conditions, and the long-term effect of this factor on DNA methylation has been examined in both animal and human studies. In rats, offspring that had experienced low levels of maternal care (i.e. reduced licking and grooming as well as archedback nursing) displayed higher cytosine methylation of NR3C1 than offspring that had received high levels of maternal care (Weaver 2007). Methylation status can also be affected by adverse experiences later in childhood: Some studies have demonstrated that adults who had experienced childhood abuse showed an altered stress response in the HPA axis, indicating an increased vulnerability to stress-related disorders such as depression (e.g. Perroud et al. 2011). Furthermore, numerous studies have reported that depression or high levels of anxiety are associated with a hyper-active HPA axis and increased cortisol secretion (e.g. Landgraf and Wigger 2002; Laryea et al. 2012). In contrast, reduced cortisol levels, which indicate a hypo-active HPA axis, have been linked with externalizing problems, such as aggressive behavior and conduct disorder (e.g. Oosterlaan et al. 2005; van de Wiel et al. 2004). To date, however, the mediating effects of DNA methylation on the glucocorticoid receptor have been

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implicated predominantly in depression and bipolar disorder (e.g. Oberlander et al. 2008; Perroud et al. 2014). As mentioned above, the activity of the HPA axis is differentially affected in externalizing disorders and depressive disorders. We hypothesize that a diverging methylation status of the NR3C1 gene might be one link between environment and mental health in terms of mediating a hyper- or hypoactive HPA axis, respectively. The aim of the present study was to investigate whether the methylation status of CpG sites at the NR3C1 locus was differentially affected in subjects with externalizing and depressive disorders, as compared to healthy controls. To enable comparisons with previous findings on NR3C1 methylation, we focused on the NR3C1 promoter/ exon 1F, and analyzed previously reported methylation sites in the above mentioned NGFI-A binding site.

Methods Sample The present investigation was conducted within the framework of the Mannheim Study of Children at Risk (MARC). This is an ongoing epidemiological cohort study of the longterm outcome of early risk factors which commenced in 1986 [for details see (Laucht et al. 1997, 2000)]. The initial sample consisted of 384 subjects of predominantly European descent ([99.0 %) born between 1986 and 1988. Infants were recruited from two obstetric and six children’s hospitals in the Rhine-Neckar Region of Germany. Participants were included consecutively into the sample according to a twofactorial design in order to enrich and to control the risk status of the sample [factor 1: the degree of obstetric complications; factor 2: the degree of psychosocial adversity (Laucht et al. 1997, 2000)]. To control for the confounding effects of family environment, only firstborn children with singleton births and German-speaking parents were included. Furthermore, children with severe physical handicaps, obvious genetic defects, or metabolic diseases were excluded. Assessments were conducted at the age of 3 months, and then at 4.5, 8, 11, 15, and 19 years. Of the initial sample, 18 (4.7 %) of participants were excluded due to the presence of a severe handicap (IQ or MQ \ 70 or neurological disorder), and 39 (10.2 %) dropped-out of the study. The present sample comprised 275 of the subjects from the initial MARC cohort (127 males, 148 females) who had agreed to participate in the 19-year assessment. For all 275 participants, blood samples from the 19-year assessment and complete data on all other relevant variables were available. The study was approved by the ethics committee of the University of Heidelberg and written informed consent was obtained from all participants.

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Assessments Early psychosocial adversity was measured according to an ‘enriched’ index, as proposed by Rutter and Quinton (Rutter and Quinton 1977). These data were derived from a standardized parent interview, which had been conducted at the initial 3-month MARC assessment. The enriched index measures the presence of 11 adverse family factors during the 1 year period before birth. These cover characteristics of: (i) the parents (e.g., psychiatric disorder, low educational level); (ii) the partnership (e.g. marital discord, early parenthood); and (iii) the family environment (e.g., poor social integration, overcrowding). At ages 4.5, 8, and 11 years, psychiatric diagnoses were assigned on the basis of the Mannheim Parent Interview (MEI (Esser et al. 1989)). The MEI is a standardized German-language structured interview adapted from Rutter’s parent interviews (Cox and Rutter 1985), which has been modified to include all symptoms of the major ICD10 diagnoses. Research has demonstrated that the MEI is a sensitive measure of psychological disorder in children (Esser et al. 1990; Laucht et al. 2001). Symptoms occurring before the age of 4.5 years were considered tentative, and were not classified as features of a depressive or externalizing disorder. At age 15 years, ICD-10 psychiatric diagnoses were assigned using the Schedule for Affective Disorders and Schizophrenia in School Age Children K-SADS (Ambrosini 2000) (German version (Delmo et al. 2000)). The K-SADS is a widely used structured diagnostic interview conducted independently with parents and adolescents, for which substantial reliability and validity data are available. At age 19 years, the Structured Clinical Interview for DSM-IV (SCID APA, 1994; German version (Wittchen et al. 1997)) was administered and ICD-10 diagnoses were derived. For the purposes of the present investigation, participants were divided into two diagnostic groups according to the presence between the ages of 4.5 and 19 years of any depressive disorder (ICD-10 codes F32.x, F33.x, F34.1, F43.2 or F92.0), or any externalizing disorder (F90.x, F91.x, F60.2) with no current or past diagnosis of depressive disorder. In addition, a healthy control group was established comprising participants with no current or past psychiatric disorder. Participants with a history of any other psychiatric disorder were excluded from the present analyses. To determine smoking status, the 15 and 19 years assessments included the completion of a substance use inventory (Mu¨ller and Abbet 1991). At the age of 19 years, an Alcohol Timeline Followback Interview (Sobell et al. 2003) was used to assess the total amount of alcohol drunk during the past month. The total amount of alcohol was

adjusted for the individual’s body weight to control for differences due to the participant’s physical constitution. Additionally, participants were asked if they had consumed cannabis during the last year and whether they currently were on psychopharmacologic medication. Methylation analyses Ethylenediaminetetraacetic acid blood samples were obtained from all participants at the age of 19 years (mean age = 19.23 years, SD = 0.29). Automated genomic DNA extraction was performed using the chemagic Magnetic Separation Module I (Chemagen Biopolymer-Technologie AG; Baesweiler; Germany). Genomic DNA samples (500 ng) were bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany), and stored at -20 °C until analysis. To allow comparability with the results of earlier studies, a 405 base pair fragment of the NR3C1 Exon 1F (NT_029289.12) was selected for analysis. This was obtained via PCR amplification (HotStar Taq DNA Polymerase, Qiagen, Hilden, Germany) of 2.5 ll bisulfitetreated DNA (Fig. 1) with the forward primer 50 TTTTTAATTTTTTAGGAAAAAGGGTGG-30 (bisulfitetreated sequence, sense) and the reverse primer 50 CCCTAAAACCTCCCCAAAAAAC-30 (bisulfite-treated, antisense). A biotin was located at the 50 end of the reverse primer (Eurofins/MWG/Operon, Ebersberg, Germany), this served for purifying the PCR product. This fragment included all 11 of the previously implicated CpG sites (McGowan et al. 2009; Oberlander et al. 2008). Cycling parameters were as follows: 95 °C for 15 min; 50 cycles respectively of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s; and a final extension of 10 min at 72 °C. PCR products were pyrosequenced using the PyroMark Q24 system (Qiagen, Hilden, Germany), and the primers 50 GTTAAGAGGGTTAT-30 (CpG 1–6) and 50 -GTTTTAGATTTATT-30 (CpGs 7–11). Successful amplification and specificity of the PCR products were checked on an agarose gel. Processing of the PCR amplicons for the pyrosequencing analysis was then performed in accordance with the manufacturer’s protocol (Qiagen, Hilden, Germany). The percentage of methylation at each CpG site was quantified using Pyro Q-CpG software version 2.0.6 (Qiagen, Hilden, Germany). The internal quality control procedure of the pyrosequencing software indicated that more than 10 % of the methylation values for site 3 were not reliable, even after repetition of the analysis. Site 3 was therefore excluded from further analysis. For the remaining 10 methylation sites and 219 included participants (i.e. 2190 values), 26 values were classified as not reliable by the software and were replaced by the mean.

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Fig. 1 Amplified and bisulfite-treated sequence of the NR3C1 gene. NCBI Reference Sequence NT_029289.12, 3,950,277–3,950,681, sense strand. Italics PCR primer; underlined forward primer for

pyrosequencing; bold methylation sites; methylation sites 1–6 from Oberlander et al. (2008) and sites 2–11 from McGowan et al. (McGowan et al. 2009)

Statistical analysis

disorder) between the ages of 4.5 and 19 years and were therefore excluded from the analyses. Among participants with a depressive disorder, 16 (59.3 %) had an additional current or past diagnosis of an externalizing disorder. The diagnostic groups significantly differed with respect to several characteristics (see Table 1): Significantly more males than females were assigned a lifetime diagnosis of externalizing disorder (v2 = 10.22, p = 0.001). In contrast, lifetime depressive disorder was significantly more prevalent among females (v2 = 7.74, p = 0.005). Significant group differences were observed for the early psychosocial adversity score (F(2,216) = 21.17, p \ 0.001). Individuals with a lifetime diagnosis of externalizing disorder or depressive disorder had experienced higher early psychosocial adversity than the healthy control group. In addition, participants with a lifetime diagnosis of externalizing disorder or depressive disorder reported a significantly increased probability of daily smoking and a significantly higher prevalence of cannabis use compared to healthy controls (externalizing disorder v2 = 14.18, p \ 0.001 and v2 = 6.14, p = 0.013; depressive disorder v2 = 10.86, p \ 0.001 and v2 = 7.45, p = 0.006). Only participants with a lifetime diagnosis of externalizing disorder reported a significantly higher use of alcohol in comparison to healthy controls (p = 0.012), but not in comparison to participants with a lifetime diagnosis of depressive disorder (p = 0.527). As to be expected, individuals with a lifetime diagnosis of a depressive disorder had a significantly higher probability of current intake of psychopharmacologic medication when compared to healthy controls (Fisher’s exact test, p = 0.001).

To examine differences between the diagnostic groups in terms of sex, age, early psychosocial adversity, smoking status, alcohol use, cannabis use, and intake of psychopharmacologic medication, v2 tests, Fisher’s exact tests and analyses of variance followed by simple contrasts were conducted as appropriate. Analyses of covariance (ANCOVAs) followed by simple contrasts were used to investigate the effect of lifetime diagnosis on NR3C1 methylation status. Sex, age, early psychosocial adversity, smoking status, and use of alcohol, cannabis and psychopharmacologic medication were included as covariates. Power considerations When planning the study, no information about NR3C1 methylation in externalizing disorders as compared to healthy controls was available. Furthermore, to the best of our knowledge very few studies directly compared patients with a depressive disorder to healthy controls with regard to the methylation of the NR3C1 gene. Only recently, in a study conducted by Na and colleagues (Na et al. 2014) patients with a depressive disorder were contrasted with healthy controls regarding NR3C1 methylation. The authors reported significantly lower methylation in depressive patients at two CpG sites, with a medium to large effect size (Cohen’s d: 0.6). Hence, when expecting a comparable effect size for group effects in the present study (f = 0.325), the available sample size was considered adequate for maintaining an adequate power level of 0.8.

Methylation level

Results Group characteristics Methylation status was determined at a mean age of 19.23 years (SD 0.29). A total of 56 of the 275 participants fulfilled the diagnostic criteria for a psychiatric disorder other than depressive or externalizing disorder (i.e. anxiety

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For lifetime psychiatric diagnosis a significant association with average NR3C1 methylation was found (F(2, 209) = 6.820, p = 0.001, Table 2). The externalizing disorders group had lower NR3C1 methylation levels compared to the depressive disorder group (p = 0. 009), and healthy controls (p = 0.001). No difference in methylation status was observed between the two latter groups (p = 0. 738)

Behav Genet Table 1 Sample characteristics

Age (years): M (SD)

LDD (N = 27)

LED (N = 68)

19.30 (0.43)

19.20 (0.24)

4 (14.8)a

Males: N (%) Early psychosocial adversity: M (SD) Daily smokers: N (%) Cannabis use during the past year: N (%)

19.22 (0.32)

46 (67.6)a,b

3.18 (2.44)a

2.67 (2.22)a

a

a

14 (51.9)

Amount of alcohol drunk during the past month, (g/kg body weight): M (SD)

32 (47.1)

4.92 (7.38)a

4.12 (6.55) 10 (37.0)a

20 (29.4)a

4 (14.8)a

2 (2.9)

Current psychopharmacologic medication: N (%)

HC (N = 124)

54 (43.7) 1.27 (1.66) 26 (21.0) 2.81 (3.95) 18 (14.5) 0

M mean, SD standard deviation, LDD lifetime depressive disorder, LED lifetime externalizing disorder, HC healthy controls Significantly different from

a

healthy controls,

Table 2 NR3C1 methylation at different CpG sites according to diagnostic group

b

lifetime depressive disorder

LDD (N = 27)

LED (N = 68)

HC (N = 124)

p

Site 1

2.12 (0.66)

1.83 (0.69)

2.06 (0.66)

0.108

Site 2

12.74 (2.77)

12.15 (2.66)

12.74 (2.30)

0.285

Site 4

5.26 (1.37)

4.92 (1.14)

0.024

a,b

4.58 (1.23)b

Site 5

6.71 (1.66)

5.72 (1.66)

6.39 (1.52)

0.009

Site 6

1.43 (0.38)

1.06 (0.46)a,b

1.25 (0.39)

0.001

Site 7

2.30 (0.71)

1.97 (0.70)a,b

2.35 (0.79)

0.004

Site 8

1.95 (0.47)

1.70 (0.59)a

1.99 (0.56)

0.004

Site 9

2.32 (0.78)

2.12 (0.74)a

2.38 (0.80)

0.044

Site 10

2.71 (0.95)

2.37 (0.73)a

2.71 (0.92)

0.016

4.28 (1.29) 4.11 (0.71)

0.007 0.001

Site 11 Mean over sites 1–11

4.39 (1.25) 4.18 (0.74)

a,b

3.75 (1.17) 3.73 (0.83)a,b

p values refer to the ANOVA group effect. Results are adjusted for sex, age, early psychosocial adversity, smoking status, and use of alcohol, cannabis and psychopharmacologic medication. Data are reported as the mean (SD) LDD lifetime depressive disorder, LED lifetime externalizing disorder, HC healthy controls, significantly different from a healthy controls, b lifetime depressive disorder

(Fig. 2). In the analysis of the single CpG sites, a significant effect on NR3C1 methylation at age 19 years was observed for eight of the ten sites (Table 2).

Discussion

Fig. 2 Means and standard errors of methylation in the CpG sites of the NR3C1 gene according to diagnostic group. LDD lifetime depressive disorder (N = 27), LED lifetime externalizing disorder (N = 68); HC Healthy controls (N = 124); *p \ 0.05

The present investigation was motivated by the observation that NR3C1 methylation findings in humans parallel those reported in experimental animals (McGowan et al. 2009; Weaver 2009), and the fact that these effects are not limited to brain tissues but are also present in the blood as an accessible peripheral proxy (Perroud et al. 2011). Using data collected over a 19 year period within the MARC epidemiological cohort study, the present investigation revealed that a lifetime diagnosis of an externalizing disorder (i.e. conduct disorder or hyperkinetic disorder) was associated with a significantly lower average methylation

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status across the sites of the NR3C1 promoter/exon 1F than was the case in healthy controls and individuals with a lifetime history of a depressive disorder. Since the various NR3C1 promoters/first exons have distinct CpG sites (Turner et al. 2008), comparability between NR3C1 studies is confined to those which analyze the same 50 exonic region. Over the past decade, numerous studies have generated evidence for altered methylation of NR3C1 promoter/exon 1F in a variety of phenotypes of relevance to psychiatric disorders. These studies have included investigations of the effects on NR3C1 promoter/ exon 1F methylation of: childhood maltreatment in borderline personality disorder (Perroud et al. 2011); traumatic events in bipolar disorder (Perroud et al. 2014); a history of childhood abuse in suicide victims (McGowan et al. 2009); intimate partner violence during pregnancy in adolescents (Radtke et al. 2011); prenatal maternal mood in infants (Oberlander et al. 2008); childhood maltreatment or adversity in healthy adults (Tyrka et al. 2012); and prenatal maternal stress in newborns (Mulligan et al. 2012). Several of these studies focused specifically on the consequences of early life stress, such as prenatal stress, inadequate maternal care, and childhood maltreatment (Mulligan et al. 2012; Oberlander et al. 2008; Tyrka et al. 2012; Weaver 2009; Weaver et al. 2004). These studies were based on the hypothesis that individuals with higher methylation exhibit altered stress responses and increased susceptibility to psychiatric disorder, suggesting that the down regulation of NR3C1 expression mediates susceptibility to these conditions. The present study demonstrated the opposite effect in subjects with externalizing disorders, since in these individuals, NR3C1 promoter/exon 1F methylation was significantly reduced compared to healthy controls and individuals with a lifetime history of a depressive disorder. Previous studies have demonstrated a hypo-active HPA axis in subjects with externalizing disorders (Gowin et al. 2013). We now hypothesize that this effect might be caused by reduced methylation of NR3C1 promoter/exon 1F. In contrast, no significant difference was found between individuals with a lifetime diagnosis of a depressive disorder and those with no history of psychiatric disorder. This finding could support the hypothesis of previous authors that depression might not be associated with increased methylation in the NR3C1 gene (Alt et al. 2010). On the other hand previously reported contradictory results on methylation in the glucocorticoid receptor in association with depression could also be due to the overall small methylation level in the glucocorticoid receptor itself. However, depressive disorders may be associated with methylation effects which were—for several possible reasons—not detected in the present analyses. Firstly, the sample sizes may have been too small to detect a

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significant difference. Secondly, adolescents and young adults may develop differing depressive disorder subtypes compared to older adults, which may result in a different methylation status. For example, a previous study of adults with chronic major depression showed that most subjects had no history of depression during adolescence (Klein et al. 1999). Moreover, depressed adolescents have been shown to differ from depressed adults in terms of several neurobiological correlates, such as basal cortisol secretion and response to serotonergic probes, as well as in their response to treatment, e.g. with tricyclic antidepressants (Kaufman et al. 2001). Furthermore, research has shown that when compared to individuals with adult onset, depressed subjects with adolescent onset were more likely to have a history of unique childhood risk factors, such as neurodevelopmental problems; psychopathology and instability in their family of origin; and behavioral problems, in particular those associated with the externalizing spectrum, such as antisocial and hyperactive behavior (Jaffee et al. 2002). Another feature specific to adolescent onset is high comorbidity with other psychiatric disorders, in particular conduct disorder (Angold et al. 1999). This also applies to the present study, where a high percentage of participants with depressive disorders also fulfilled the criteria of a past or current externalizing disorder. This line of evidence suggests that depression in young adulthood may represent a heterogeneous phenotype, which should be differentiated into developmentally specific subtypes, with the adolescent-onset subtype being characterized by a particularly high psychosocial load and a higher prevalence of externalizing disorders. Limitations Limitations of the study are as follows: Firstly, the sample size was small for an epigenetic association investigation and association studies with small samples are prone to false positive results. Our results therefore require validation in larger, independent samples. Secondly, the possibility that sex differences may have contributed to the differential levels of methylation cannot be excluded, since the externalizing disorders group was mainly comprised of males (67.6 %), and previous reports have shown hypermethylation of the NR3C1 gene in hippocampal DNA in female rats (Kosten et al. 2013). However, we controlled for sex in all respective analyses to ensure that the potential impact of a sex effect is likely to be minimal. Thirdly, due to the naturally small methylation levels in the glucocorticoid receptor gene it might be useful to conduct multiple measuring of each sample to minimize measurement errors and to ensure valid results. However, as most methylation sites show a consistently reduced methylation level in participants with a lifetime externalizing disorder

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(Table 2), we are confident that our results are at least a strong indicator for a differential effect in externalizing disorders compared to depressive disorders. Fourthly, the present study is unable to determine whether methylation status is a cause or a consequence of externalizing disorders. In conclusion, the present study is the first to provide evidence that decreased NR3C1 promoter/exon 1F methylation is implicated in externalizing disorders during childhood and adolescence. Further research is warranted to identify which specific life events are responsible for this decreased methylation effect. Acknowledgments This research was supported by the German Ministry of Education and Research within the context of the National Genome Research Network plus (NGFNplus) and the MooDS-Net (Grant 01GS08147 to MR). AH and VN received support from the Olympia-Morata-Program of the University of Heidelberg. Conflict of interest

The authors declare no conflicts of interest.

Human and Animal Rights and Informed Consent All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study.

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Alterations of Glucocorticoid Receptor Gene Methylation in Externalizing Disorders During Childhood and Adolescence.

Epigenetic modulations are a hypothesized link between environmental factors and the development of psychiatric disorders. Research has suggested that...
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