Journal of Psychiatric Research 46 (2012) 38e43 Contents lists available at SciVerse ScienceDirect Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires The impact of adverse life events and the serotonin transporter gene promoter polymorphism on the development of eating disorder symptoms Kirsti Akkermann a, Kadri Kaasik a, Evelyn Kiive a, Niklas Nordquist b, Lars Oreland b, Jaanus Harro a, * a b Department of Psychology, Estonian Centre of Behavioural and Health Sciences, University of Tartu, Tiigi 78, Tartu 50410, Estonia Department of Neuroscience, Pharmacology, University of Uppsala, Sweden a r t i c l e i n f o a b s t r a c t Article history: Received 12 April 2011 Received in revised form 29 September 2011 Accepted 29 September 2011 Adverse life events have been shown to predict weight fluctuations and dietary restraint, as well as eating disorders during adolescence or early adulthood. Since the s-allele carriers of the 5-HTT genelinked polymorphic region (5-HTTLPR) are biologically more reactive to stress related stimuli, we aimed to explore whether the eating disturbances are predicted by environmental stressors and moderated by the 5-HTTLPR genotype. The sample was based on the younger cohort of the Estonian Children Personality, Behaviour and Health Study and included those participating in its second and third wave. The history of stressful life events was self-reported at age 15. Data on eating behaviour and attitudes, anxiety, impulsivity and depressiveness were collected at age 18. The effect of the adverse life events on binge eating and on drive for thinness was found to be moderated by the 5-HTTLPR. Adolescent girls who at age 15 had reported a history of frequent adverse life events had elevated scores in EDI-2 Bulimia subscale at age 18 if they were carrying the s-allele. The effect of the s-allele on binge eating was even more pronounced when solely the experience of sexual abuse was considered. The interaction effect of the 5-HTTLPR and the past sexual abuse was also observed on drive for thinness. These data give further support to the idea that adverse life events in childhood may heighten susceptibility to serotonergic dysregulation following stress, and suggest that in individuals vulnerable to eating disorders this may result in disturbed eating behaviours. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: 5-HTTLPR Binge eating Drive for thinness Adverse life events Geneeenvironment interactions 1. Introduction The serotonin transporter mediates sodium-dependent presynaptic re-uptake of serotonin, thus terminating serotonergic neurotransmission. The serotonin transporter is encoded by the serotonin transporter gene (SLC6A4) on chromosome 17 (Gelernter et al., 1995). The short or s-allele in the 5-HTT gene-linked polymorphic region (5-HTTLPR) is associated with lower transcriptional activity of the promoter as compared to the long or l-allele, and has been suggested to lead to lower expression of 5-HTT mRNA, less serotonin (5-HT) binding, and less 5-HT re-uptake (Lesch et al., 1996). Human neuroimaging studies of the 5-HTT availability have not detected consistent effects of the 5-HTTLPR (Parsey et al., 2006; Shioe et al., 2003) but the allelic differences in the 5-HTTLPR have been demonstrated to affect brain volume and gray-matter density in multiple brain regions in humans (Canli et al., 2005; Pezawas et al., 2005) and non-human primates (Jedema et al., 2010). * Corresponding author. Tel.: þ372 7375911; fax: þ372 7375900. E-mail address: jaanus.harro@ut.ee (J. Harro). 0022-3956/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2011.09.013 The s-allele has been associated with trait anxiety (Lesch et al., 1996; Sen et al., 2004), affective instability (Lesch and Mössner, 1998; Steiger et al., 2005) and impulse control deficiencies (Retz et al., 2004; Anguelova et al., 2003; Baca-García et al., 2005). Individuals carrying one or two copies of the s-allele have been reported to show greater amygdala reactivity to emotion-related stimuli (Hariri et al., 2002), and stronger and longer lasting reactions to fearful stimuli (Armbruster et al., 2009). The imaging genetics studies show that the s-allele leads to reduced gray-matter volume in limbic regions (Heinz et al., 2005) and disrupted amygdala-cingulate coupling after emotional stimuli (Pezawas et al., 2005). Since Caspi et al. (2003) reported that individuals with one or two copies of the 5-HTTLPR s-allele exhibited more depressive symptoms and suicidality in relation to stressful life events than did individuals homozygous for the l-allele, research looking at the geneenvironment effect in the development of affective disorders has been growing rapidly (Cervilla et al., 2007; Wilhelm et al., 2006; Kendler et al., 2005). Although meta-analyses have not always been supportive of the interaction effect between the 5HTTLPR and adverse life events on depression (Munafo et al., 39 K. Akkermann et al. / Journal of Psychiatric Research 46 (2012) 38e43 2009; Risch et al., 2009), systematic reviews confirm this effect indicating that genotype moderates the capacity of an environmental risk to bring about mental disorders (Nugent et al., 2011; Uher and McGuffin, 2010). The differences in results may rather be caused by methodological considerations that influence the inclusion of the studies in meta-analyses, outcome measures, relative power of included studies, etc. The research looking at the geneeenvironment effects in regard to eating disorders is so far limited. There is some evidence that the onset of eating disorders is associated with adverse life events (Horesh et al., 1995; Welch et al., 1997). Adverse life events, such as physical neglect, maladaptive parenting behaviours, socioeconomic variables (poverty, limited parental education) and sexual abuse during childhood, have been shown to predict weight fluctuations and dietary restraint, as well as eating disorders during adolescense or early adulthood in a community based sample (Johnson et al., 2002). Also, an association between stressful life events with extreme weight control behaviours and binge eating has been reported in a community based sample of adolescents (Loth et al., 2008) and young adults (Smyth et al., 2008). Childhood experiences of being bullied have been reported to result in eating-disordered behaviours (Wade et al., 2007) and teasing about appearance in increased body dissatisfaction (Sweetingham and Waller, 2008). One of the most discussed topics in the development of eating disorders is the role of sexual abuse. Welch and Fairburn (1994) have reported sexual abuse to be significantly more common in bulimic patients when compared to controls, but found no difference between bulimic patients and other psychiatric patients. Jacobi et al. (2004) concluded in their review that there is some evidence that anorexia and bulimia patients experience more adverse life events before the onset of the eating disorder, although the evidence is not as consistent as it is for the role of sexual abuse. While some studies have reported an association between the 5-HTTLPR s-allele and eating disorders (Di Bella et al., 2000; Fumeron et al., 2001), most studies have found no allelic differences in the 5-HTTLPR in eating disorder patients (Hinney et al., 1997; Sundaramurthy et al., 2000; Lauzurica et al., 2003; Monteleone et al., 2006; Steiger et al., 2005; Racine et al., 2009). Recently we reported that while the 5-HTTLPR genotype does not predict symptoms of binge eating, the s-allele, and especially the s/s genotype, increases the risk for affective instability and symptom severity in general population (Akkermann et al., 2010). Data from patients with affective disorders suggest that s-allele carriers are more sensitive and reactive to life circumstances. Eating disorders are often comorbid with affective disorders (Mangweth et al., 2003) and eating disorder patients, carrying the s-allele, have shown to display higher psychiatric comorbidity like major depressive disorder, anxiety disorders, and substance abuse (Richardson et al., 2008). Thus, it could be hypothesised that the ability of adverse life events to elicit disturbed eating behaviour is more pronounced in carriers of the s-allele. To our knowledge no study has so far addressed the role of adverse life events and the 5-HTTLPR together in bringing about symptoms of eating disorders. Thus, in the present study we aimed to examine the gene  environment effect of the 5-HTTLPR and adverse life events on disturbed eating behaviours in a population-representative sample of adolescent girls. Since the literature suggests that traumatic experiences involving the element of interpersonal harm (e.g., sexual abuse, physical assault) are associated with higher risk for psychiatric disturbances we also explored separately the interactive effect of sexual, physical and emotional abuse and 5-HTTLPR on symptoms of eating disorders. 2. Materials and methods 2.1. Participants The sample was based on younger cohort of the European Youth Heart Study (EYHS) conducted in Estonia in 1998/1999, which was incorporated into the longitudinal Estonian Children Personality, Behaviour and Health Study (ECPBHS). Sample formation has been explained and described in more detail elsewhere (Harro et al., 2001, 2009). In brief, this is a population-representative age cohort of urban and rural girls and boys living in at the time of sampling in one county. All schools of Tartu City and County, Estonia, which agreed to participate in the study, were included into the sampling using the probability proportional to the number of children of the respective age group in the school. Totally, 583 children from 25 schools were participating in the first wave of the study. This was 80% of the eligible population. The data presented in the current study was gathered during the second and third follow- up in years 2004 and 2007, where totally 83% (n ¼ 483) and 78% (n ¼ 453) of the original sample was recruited, respectively. Only the data of girls has been used since the prevalence of disturbed eating behaviour in boys was low. The data of 261 girls (mean age 14.8  0.5) and 252 girls (mean age 17.8  0.5) from the second and third wave, respectively, were analysed. Blood samples and self-reported data about their eating behaviour, adverse life events, depression and anxiety levels were collected in the laboratory among other measurements (Table 1). Adolescents and their parents gave their written informed consent in all study waves. The study was approved by the Ethics Review Committee on Human Research of the University of Tartu, Estonia. The study was carried out in accordance with the Declaration of Helsinki. 2.2. Measures The history of adverse life events was self-reported at age 15. The list of stressful life events was formed of 30 adverse experiences, including parental death and divorce/separation, living in institution or under foster care, unemployed parent, parental alcoholism, poor parental care, poverty, poor living conditions, poor health, accidents and traumas, school bullying, recurrent physical punishment, physical, sexual and emotional abuse in- and outside the family, severe burden/serious concerns, attempted suicide, attempted rape, leaving home for several days without telling anyone, and committed suicide or suicide attempt of a close relative. The events were recorded dichotomously, present or not present during lifetime, and were then counted to form the total Table 1 Descriptive and clinical data (mean scores  SD) of the participants. Age at the 1st assessment Age at the 2nd assessment 14.8 (0.52) 17.8 (0.52) EDI-2 Bulimia Drive for thinness 4.9 (5.02) 9.6 (7.67) STAI State anxiety Trait anxiety 33.2 (8.97) 41.7 (9.39) MADRS BIS-11 8.5 (5.73) 62.5 (5.49) 5-HTTLPR l/l s/l s/s n ¼ 103 n ¼ 110 n ¼ 26 40 K. Akkermann et al. / Journal of Psychiatric Research 46 (2012) 38e43 number of adverse life events. On the bases of the percentile distribution (25e50e25) the subjects were divided into groups with minimal (0e1 events), moderate (2e5 events) and frequent (6e18 events) history of adverse life events. Data on eating behaviour and attitudes, anxiety, impulsivity and depressiveness was collected at age 18. Eating Disorders Inventory2 (EDI-2) (Garner, 1991), Estonian version (Podar et al., 1999) two subscales e Drive for thinness and Bulimia e were used to assess eating behaviour and attitudes. The Drive for thinness subscale measures concern and preoccupation with dieting and weight gain, the Bulimia subscale measures the tendency to think about and engage in episodes of binge eating. These subscales have been shown to be most directly related to eating-disordered behaviour (Hurley et al., 1990). Anxiety was measured by Estonian version of State and Trait Anxiety Inventory (STAI) (Spielberger et al., 1983; Kreegipuu, 1997). Depression ratings were collected by means of self-report version of MontgomeryeÅsberg Depression Rating Scale (MADRS) (Montgomery and Åsberg, 1979), using the Estonian version (Kiive et al., 2004). Estonian version of Barratt Impulsiveness Scale (BIS-11) (Patton et al., 1995; Paaver et al., 2007) was used to measure impulsivity. 2.3. Genotyping The alleles at the 5-HTTLPR locus were amplified from genomic DNA using polymerase chain reaction (PCR). The polymorphic region was amplified using the primers; 5-HTTLPR-F: CAA CCT CCC AGC AAC TCC CTG TA, 5-HTTLPR-R: GAG GGA CTG AGC TGG ACA ACC AC, where the forward primer was labelled with a 50 -FAM. Reagents and conditions for the PCR reaction were: 1 PCR buffer (Perkin Elmer, AmpliTaq Gold buffer II), 200 mM dNTP with 50% of dGTP replaced with 7-deaza-dGTP, 2 mM MgCl2, 1 mM of each primer, 1 U Taq polymerase (Perkin Elmer, AmpliTaq Gold), and 20 ng genomic DNA, in a total reaction volume of 10 mL. The reaction started with 10 min at 95  C, followed by 35 cycles with 30 s at 95  C, 30 s at 59  C, 30 s at 72  C, and ended with 10 min at 72  C. PCR products were then run on an ABI 3100 Genetic analyzer (Applied Biosystems), and scored using the software GeneMapper 1.5 (SoftGenetics). All genotypes were manually checked on chromatograms to detect inconsistencies, and, where needed, amplified and scored the second time. Genotype frequencies were distributed in HardyeWeinberg equilibrium. Genotypic data were classified into two groups, l/l homozygotes (n ¼ 103) vs s-allele carriers (n ¼ 136). 3. Results The 5-HTTLPR allelic variation itself was not associated with either EDI-2 Bulimia, F(1, 207) ¼ 0.41, p ¼ 0.52 or Drive for thinness scores, F(1, 207) ¼ 1.00, p ¼ 0.32. Also, no significant differences between the s-allele carriers and l/l homozygotes concerning the number of adverse life events were observed, F(1, 234) ¼ 1.13, p ¼ 0.29. However, there was an interaction effect of the 5-HTTLPR and adverse life events on Bulimia scores, F(2, 200) ¼ 3.29, p ¼ 0.04. Thus, the more negative life events the s-allele carriers had experienced by age 15, the higher EDI-2 Bulimia scores they reported at age 18 (Fig. 1). Carriers of the s-allele, who had history of frequent adverse life events, exhibited higher scores on the Bulimia subscale as compared to both the s-allele carriers and l/l homozygotes with history of minimal adverse life events (p ¼ 0.02 and p ¼ 0.001, respectively). Similar tendencies of a difference were observed between the s-allele carriers with history of frequent adverse life events and individuals reporting the history of moderate number of adverse life events, but statistical significance was below the conventional level (p ¼ 0.06 and p ¼ 0.09, respectively for comparisons with l/l homozygotes and s-allele carriers). Likewise, the s-allele carriers with frequent adverse life events tended to score higher on the Bulimia subscale as compared to l/l homozygotes with history of frequent adverse life events (p ¼ 0.08). The interaction effect explained 6% (R2 ¼ 0.06, p ¼ 0.02) of the total variance in Bulimia scores. Importantly, this interaction effect remained significant even after the co-varying effects of depression and state and trait anxiety were controlled for (p ¼ 0.02, 0.01, 0.03 respectively) and nearly significant after the co-varying effect of impulsivity was controlled for (p ¼ 0.06). The effect of the s-allele on Bulimia scores was even more pronounced when solely the experience of sexual abuse was considered, F(1, 197) ¼ 9.64, p ¼ 0.002 (Fig. 2B), explaining 9% (R2 ¼ 0.09, p < 0.001) of the total variance. The interaction effect remained significant after controlling for the co-varying effect of impulsivity (p ¼ 0.001). The significance of the interaction effect on Bulimia scores was missed when only the history of physical abuse was included to the 2.4. Statistical analyses Statistical analyses were carried out using Statistica version 7.0 software. First we tested whether the 5-HTTLPR allelic variation itself is associated with EDI-2 subscales and whether there are allelic differences in the frequency of adverse life events. Associations between the test scores and the 5-HTTLPR genotype were assessed by one-way analysis of variance (ANOVA). Further we proceeded looking at the interaction effects of the 5-HTTLPR and adverse life events on EDI-2 Bulimia and Drive for Thinness subscales. Interaction effects between the genotype  group were assessed by two-way ANOVA. For comparison of the groups Fischer LSD post hoc analyses was used. Since the 5-HTTLPR has been shown to be associated with depression, anxiety and impulsive behaviour we used ANCOVA to control for co-varying effects entering the total scores of depression, anxiety and impulsivity independantly as continuous covariates. Finally we perfomed the analyses to test whether sexual, physical and emotional abuse together with the 5-HTTLPR predicts disturbed eating as measured by EDI-2. Fig. 1. The influence of adverse life events and the 5-HTTLPR genotype on binge eating (presented as EDI-2 Bulimia z-scores). Vertical bars denote 0.95 confidence interval K. Akkermann et al. / Journal of Psychiatric Research 46 (2012) 38e43 model, F(1, 199) ¼ 3.22, p ¼ 0.08, and was clearly not present when only the experience of emotional abuse was included, F(1, 199) ¼ 0.62, p ¼ 0.43. The interaction effect of the 5-HTTLPR and the experience of sexual abuse was observed also on Drive for thinness scores, F(1,197) ¼ 4.69, p ¼ 0.03 (Fig. 2A). This interaction effect remained significant even after the co-varying effect of impulsivity was controlled for (p ¼ 0.04). Carriers of the s-allele, who reported the history of sexual abuse, exhibited higher scores on Drive for thinness subscale as compared to the s-allele carriers and l/l homozygotes who did not report the sexual abuse (p ¼ 0.0004 and p ¼ 0.005, respectively). Similar tendency of a difference was observed when compared to the l/l homozygotes with history of sexual abuse but statistical significance was below the conventional level (p ¼ 0.07). Fig. 2. The influence of sexual abuse and the 5-HTTLPR genotype on drive for thinness (presented as EDI-2 Drive for thinness z-scores) (A) and binge eating (presented as EDI-2 Bulimia z-scores) (B). Vertical bars denote 0.95 confidence intervals. 41 4. Discussion Psychosocial stressors accentuate genetic vulnerability to psychopathology in adolescents (O’Connor et al., 2003; Heider et al., 2008). We report in the present paper that the effect of adverse life events on binge eating was moderated by the 5HTTLPR. Individuals with the s-allele, who at age 15 reported history of frequent adverse life events, had elevated scores in EDI-2 Bulimia subscale at age 18. The effect of the s-allele on binge eating was even more pronounced when solely the experience of sexual abuse was considered. The interaction effect of the 5-HTTLPR and the past sexual abuse was also observed on drive for thinness. Steiger et al. (2004) reported that bulimia nervosa patients with reduced platelet paroxetine binding (as compared to those with higher binding) had more likely experienced childhood sexual abuse, indicating a coincidence of traumatic experiences with hyposerotonergic tendencies in adult women with eating disorders. The authors argued that childhood abuse may contribute to longlasting sensitivities of the 5-HT system and in that case such effects might heighten susceptibility to serotonergic dysregulation following stress or negative affects. Depue and Collins (1999) suggest that individuals low in 5-HT activity are hypersensitive to sensory input so that even low-intensity stimuli may elicit a response. Consequently, each new input serves as an emotioneliciting stimulus after another, which finally manifests in emotional lability and impulsive behaviour. In line with this, trauma in eating disorder patients has been associated with greater impulsiveness and presence of borderline personality traits (Basurte et al., 2004). Low 5-HT activity in adulthood could be brought about by high levels of 5-HT, the result of the low rate of 5HT re-uptake linked to the s-allele of the 5-HTT, which reduce the development of brain 5-HT structures (Whitaker-Azmitia, 2005; Nordquist and Oreland, 2010). Several lines of evidence suggest that the CNS serotonin system plays a role in regulating HPA axis activity. Serotonergic systems can either facilitate or inhibit HPA axis activity and stress related physiological or behavioural responses (Lowry, 2002). Functional studies of the raphe nucleus and serotonergic projections arising from it have suggested that serotonergic systems within the raphe nucleus contribute to resistance, tolerance and coping responses with the acute or chronic stress (Deakin, 1996). 5-HTT gene knockout mice have been found to exhibit increased HPA axis in response to acute stress (Li et al., 1999). Recently it was reported that girls homozygous for the s-allele produce higher and prolonged levels of cortisol in response to stress than l-allele carriers, indicating that the 5-HTT gene variation affects HPA axis activity (Gotlib et al., 2008). Similar lines of evidence come from animal research. Among infant rhesus macaques the s-allele carriers had higher levels of adrenocorticotropic hormone in response to maternal separation than did l/l homozygotes (Barr et al., 2004; McCormac et al., 2009). It has been suggested that abnormalities in the response mechanisms to stress and in the HPA axis functioning could explain the association between traumatic childhood events and eating disorders (Basurte et al., 2004). While the s-allele carriers may be biologically more reactive to stress related stimuli, then in individuals vulnerable to eating disturbances this may manifest in more severe binge eating. Also, the activation of the HPA axis under conditions of chronic stress tends to heighten the more prolonged central actions of glycocorticoids in the orexigenic appetite centers (Kyrou et al., 2006). So far little is known about the specific gene-environmental effects on bringing about eating disorder symptoms. However, the effect of the adverse life events on the development of psychopathology is not specific to eating disorders but also to depression, anxiety and stress related disorders. Eating disorders 42 K. Akkermann et al. / Journal of Psychiatric Research 46 (2012) 38e43 are often comorbid with the latter but in clinical practice we can also see eating disturbances in affective and anxiety disorders. Thus assessment of the gene-environment interactions helps us to identify possible intermediate phenotypes and hopefully identify causal processes through which the environment increases the risk for the disturbed eating behaviours. Serotonin is involved in a broad range of biological, physiological and behavioural functions such as motor activity, eating, sleep and thermoregulation, cardiovascular and respiratory activity, as well as the modulation of affective states (Lucki, 1998). Environmental adversities in interaction with the 5-HTTLPR affect also broad range of functions and is probably not specific to eating disturbances. Animal studies indicate that 5-HTT knockout mice exhibit a reduction in 5-HT1A receptor binding in several brain areas (Li et al., 1999) and increase of binding density of 5-HT2A receptors in the amygdala (Li et al., 2003) which together may explain the heightened stress vulnerability in these mice (Adamec et al., 2008). Alterations in 5-HT1A and 5-HT2A receptor bindings have been reported in eating disorders patients (Bailer et al., 2004, 2005). Thus possible synergistic effects of the serotonin system following exposure to environmental adversities may heighten affective instability and affect regulation difficulties in sallele carriers which in turn may manifest in increased binge eating. Recently we reported that the effect of BDNF Val66Met polymorphism on binge eating in adolescent girls is dependent on severe food restriction (Akkermann et al., 2011). The BDNF Metallele carriers who practiced the most extreme weight control behaviours like reducing their meal frequency and/or starvation, which was also validated by their reduced caloric intake, were more engaged in binge eating. This effect was not observed on drive for thinness. Thus, there may be specific environmental effects acting on certain genotypes and bringing about specific disturbances in eating behaviours but the research is still limited in this field. To conclude, the inconsistent results of the association between the 5-HTTLPR and eating disorder symptoms may be because participant`s history of adverse life events has not been considered in these studies. The results of the present study must be considered in the context of the limitations. We have been analysing only one genetic marker, though it is unlikely that the 5-HTTLPR interacts with environmental factors independently of other genes. Gene  gene  environment interactions would most likely explain even larger variability in symptoms of eating disorders in response to adverse life events. Also, these findings need to be replicated possibly in larger samples and ideally objective methods of environmental adversity should be used. Conflict of interest All authors report no conflict of interest. Contribution Kirsti Akkermann was responsible for the study design, collecting the data, conducting statistical analysis, and preparing most of the sections of the manuscript. Kadri Kaasik contributed to the data preparation, data collection, data analysis and preparing the figures. Evelyn Kiive contributed to data preparation and the section of the manuscript related to materials and methods. Niklas Nordquist was responsible for genotyping and preparing the section of the manuscript related to genotyping. Prof. Oreland edited drafts of the manuscript. Prof. Harro was responsible for the study design, oversight of data collection and editing drafts of the manuscript. All authors have contributed to and approved the final manuscript. Financial disclosures All authors report no biomedical financial interests or potential conflicts of interest. Funding This study was supported by grants from the Estonian Ministry of Education and Science (No. 0180027), the Estonian Science Foundation (No. 6788 and 8622) and the Swedish Science Foundation (No. 3640). Funding sources had no role in writing of the present manuscript. Acknowledgements We are grateful to the participants of the ECPBHS, and to the whole ECPBHS Study Team. References Adamec R, Holmes A, Blundell J. 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