5-HTTLPR S-allele: a genetic plasticity factor regarding the effects of life events on personality?

Transcription

1 Genes, Brain and Behavior (2012) 11: doi: /j X x 5-HTTLPR S-allele: a genetic plasticity factor regarding the effects of life events on personality? Y. Kuepper,, C. Wielpuetz, N. Alexander, E. Mueller,P.Grant and J. Hennig Personality Psychology and Individual Differences, Department of Psychology, Justus-Liebig-University Giessen, Giessen, Germany, and Department of Psychology, Chair of the Biopsychology, Dresden University of Technology, Dresden, Germany *Corresponding author: Y. Kuepper, Personality Psychology and Individual Differences, Department of Psychology, Justus-Liebig-University Giessen, FRG, Otto-Behaghel-Str. 10F, D Giessen, Germany. yvonne.kuepper@psychol. uni-giessen.de The S-allele of the 5-HTTLPR has been identified as a genetic vulnerability factor, being associated with an increased risk for affective disorders and/or maladaptive traits (e.g. neuroticism), especially after exposition to negative life-events (LEs). Alternatively, it has been hypothesized that this genetic risk factor might constitute a genetic plasticity factor. That is, S-allele carriers are not only vulnerable to the negative effects of a preponderance of stressful LEs but also disproportionally benefit from a preponderance of positive environmental influences. We tested this hypothesis in 357 subjects who were genotyped for the 5-HTTLPR and provided self-reports of neuroticism, life-satisfaction and LEs. Results showed a relatively increased number of positive LEss to be associated with reduced neuroticism (men: β = 0.501, P < 0.05, women: β = 0.369, P < 0.005) and increased life satisfaction (β = 0.494, P < 0.001) within SS-homozygotes. Within SL-heterozygotes, similar tendencies were found. No associations were detected in LL-homozygotes. Extreme Group comparisons revealed a genotype LE interaction (F 2,198 = 5.593, P < 0.005), with SShomozygotes having experienced predominantly positive LEs exhibiting reduced neuroticism (women: F 1,34 = 4.764, P < 0.05; men: F 1,17 = 2.092, P = 0.17), and increased life satisfaction (F 1,53 = 4.057, P < 0.05), as compared to LL-homozygotes having experienced predominantly positive LEs. Our data support the idea that the S-allele of the 5-HTTLPR is associated with an overall increased reactivity to environmental influences, be they positive or negative in nature. These findings constitute a promising add-on to earlier data and support the plasticity hypothesis. Keywords: Gene environment interaction, 5-HTTLPR, life satisfaction, neuroticism, personality, positive life-events, stressful life-events Received 20 December 2011, revised 7 February 2012, accepted for publication 12 March 2012 Neuroticism, a personality trait being associated with an increased risk for several affective disorders, as well as its etiology has been studied for decades. One focus has been the main effects and interplay of genetic and environmental factors on neuroticism. In this context, one of the most widely studied genetic factors is the serotonin transporter-linked polymorphic region (5-HTTLPR), a functional polymorphism in the promoter region of the serotonin reuptake transporter gene [details: (Lesch et al. 1996; Nakamura et al. 2000; Wendland et al. 2006)]. While numerous molecular association studies have shown the short-allele (S-allele) to be associated with increased scores in neuroticism or associated constructs, these results have been challenged by several non-replications (Munafo et al. 2006, 2008, 2009; Schinka et al. 2004; Sen et al. 2004; Willis-Owen et al. 2005). This discrepancy has been partially resolved by showing the 5-HTTLPR genotype association with neuroticism to be modulated by environmental stimuli, e.g. traumatic or adverse life-events (LEs) [review: (Caspi et al. 2010)]. A combination of having experienced adverse LEs with being an S-allele carrier increases the risk for depression and other adverse outcomes this being especially pronounced in SS-homozygotes, while LLhomozygotes appear non-reactive to negative environmental stimuli. Even though the robustness of this interaction has been challenged by several non-replications [reviews: (Middeldorp et al. 2010; Risch et al. 2009)], this is generally interpreted as the S-allele being a genetic vulnerability factor associated with increased risk for affective disorders and maladaptive traits, e.g. neuroticism, after exposition with negative LEs (Caspi et al. 2010). Some authors have questioned whether this vulnerability hypothesis explains all data. Indeed, Belsky et al. (2009) concluded that it does not; they summarize several studies indicating that SShomozygotes benefit from the absence of negative LEs more than the LL-homozygotes even though they are also more negatively affected by negative environmental stimuli. For example, in a non-risk environment, adolescent women with the SS-genotype tend to show a reduced risk for depression compared to LL-subjects (Eley et al. 2004). Taylor et al. (2006) showed SS-subjects who had grown up in a supportive environment or had experienced predominantly positive recent LEs to exhibit the fewest depressive symptoms compared to all other groups. Therefore, the question was raised whether the S-allele is associated with an overall increased reactivity to 2012 The Authors 643 Genes, Brain and Behavior 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society

2 Kuepperetal. environmental stimuli, regardless of their valence. Thus, S-allele carriers would be more vulnerable to the negative effects of stressful LEs but would also benefit disproportionally from positive environmental influences. A recent study showed such an effect; Pluess et al. (2010) analyzed the association between positive and negative LEs within the last 6 months and neuroticism. They found an association between recent LEs and neuroticism only within the SShomozygotes. This study indicates that indeed, the S-allele might not per se be a genetic risk factor, but rather constitutes a genetic plasticity factor associated with increased reactivity to environmental stimuli regardless of their valence. It, therefore, appears promising to explore this idea further. Aims of the study We were interested in replicating and broadening the findings described above. Beyond looking at neuroticism as an indicator of maladaptive personality traits, we included an indicator of positive/health associated trait personality, choosing life satisfaction for this. Furthermore, we chose to asses positive and negative LEs throughout the entire life span rather than only within the last 6 months in order to reach a more generalizable idea of the impact of LEs on personality and its modulation by 5-HTTLPR genotype. Specifically, we tested the following questions: (1) do S-allele carriers, especially SS-homozygotes, show an increased reactivity to positive and negative LEs throughout their life span? and (2) do S-allele carriers, especially SShomozygotes, benefit more from a preponderance of positive LEs than LL-homozygotes? Materials and methods Sample characteristics We tested 357 young adults (112 men and 245 women, mean age = ± 3.1 years), all of them were students at the University of Giessen with Caucasian ethnicity. In order to ensure psychological and physical health, questionnaires and, if necessary, additional interviews were performed. All participants were informed on the study objectives and gave their written informed consent. The study was approved by the Ethics Committee of the German Psychologist Association. Genotyping DNA was extracted from buccal cells using a standard commercial extraction kit (High Pure PCR Template Preparation Kit; Roche, Mannheim, Germany) in a MagNA Pure LC System (Roche). Genotyping for the triallelic classification of the 5-HTTLPR/rs25531 was performed in our lab as described previously by Alexander et al. (2009). Genotype distribution did not deviate significantly from Hardy Weinberg equilibrium (χ 2 = 2.01, P > 0.05). Genotype frequencies and functional genotype groups are shown in Table 1. Psychometric measures Personality i.e. neuroticism and life satisfaction were assessed through self-reports, using the revised version of the Freiburg Personality Inventory (FPI-R) (Fahrenberg et al. 1984). This questionnaire is widely used in Germany and has been proven to be reliable and Table 1: Genotype frequencies (triallelic classification) and functional genotype groups Genotype (triallelic) n Frequency Functional genotype group SS SS (N = 84) SL G L G L G SL A LS (N = 164) L A L G L A L A LL (N = 109) valid in a number of studies (see Fahrenberg et al. 1984). Each scale consisted of 12 self-descriptive statements which were answered in a forced choice format (true/untrue). Neuroticism and life satisfaction scores were calculated as sum values (items answered true ) for each scale, as instructed in the manual. The life satisfaction scale of the FPI-R asseses several items capturing feelings of satisfaction and contentment with one s life such as All in all, I am absolutely content with my past life., Usually, I am very optimistic about my future. or (inverted item) I am often dissatisfied with my current living conditions. Positive and negative LEs were assessed by use of self-reports with an adapted version of the Life Experience Survey (Sarason et al. 1978). Essentially, we made two changes. Firstly, we added several events from the LEs checklist (Gray et al. 2004) and the Social Readjustment Rating Scale (Holmes et al. 1967) in order to get a more representative scope of LEs. Secondly, we used a life history calendar format which has been shown to be a reliable instrument to asses LEs in retrospective (Belli et al. 2001; Caspi et al. 2003). Beyond assessing the incidences of LEs, following the original questionnaire as designed by Sarason (1978), subjects rated each LE with respect to its valence on a five-point scale: very positive (1), positive (2), neutral (3), negative (4) and very negative (5). For each subject, the total number of experienced positive LEs was calculated by summing up the number of events which were classified as very positive or positive. Accordingly, the total number of negative LEs was calculated by summing up the number of events rated as negative or very negative. A score (relle-score) reflecting the relative number of positive and negative LEs experienced by a given subject was then calculated by subtracting the number of negative LEs from the number of positive LEs. Therefore, those subjects with a value >0 predominantly experienced positive LEs, while a score <0 indicates a preponderance of negative LEs. A full list of the LEs assessed as well as the corresponding average valence ratings and number of occurrences can be found in the Table S1. Statistics We tested for associations between the different measures assessed as well as for the effects of potential confounding variables (age and sex) using Pearson correlations, chi-square analyses and analyses of variance (ANOVAs). Outcome variables, neuroticism and life satisfaction scores were calculated as per instruction of the FPI-R manual. The relative number of positive and negative LEs (relle-score) was calculated by subtracting the number of negative LEs from the number of positive LEs. In order to test whether 5-HTTLPR genotype was a moderator of the association between the relative number of positive and negative LEs and personality, we used hierarchical regression models, entering variables in the following order: age and sex (1: men, 2: women) relle-score, 5-HTTLPR genotype (0: LaLa-Grp, 1: LS- Grp, 2: SS-Grp) interaction term relle-score 5-HTTLRP genotype. Post hoc analyses were performed using simple regression models separated by 5-HTTLPR genotype groups. In order to test whether S-allele carriers would indeed benefit disproportionately from a preponderance of positive LEs, we used 644 Genes, Brain and Behavior (2012) 11:

3 5-HTTLPR S-allele as a genetic plasticity factor an extreme group approach, only looking at subjects with a clear preponderance of positive or negative LEs; subjects with a relle score of > = 3 were included in the preponderance of positive LEs group, whereas those with relle scores of < = 3 were included in the preponderance of negative LEs group. Group comparisons were performed by means of ANOVAs with two between-subject factors [factor 1: genotype, three levels (1: SS, 2: LS, 3: LL), factor 2: LEs, 2 levels (1: preponderance of positive LEs, 2: preponderance of negative LEs)] and life satisfaction or neuroticism as dependent variables. Post hoc group comparisons were performed hypothesis-driven using oneway ANOVA. That is, the following group comparisons were calculated: (1) SS-homozygotes plus preponderance of negative LEs vs. LL-homozygotes plus preponderance of negative LEs and (2) SS-homozygotes plus preponderance of positive LEs vs. LL-homozygotes plus preponderance of positive LEs. Results Associations between assessed measures and effects of potentially confounding variables An exploratory data analysis using Pearson correlations showed a significant inverse association between neuroticism scores and life satisfaction scores (r = 65, P < 0.001). Age was not associated with either outcome variable (neuroticism: r = 0.072, P > 0.05, life satisfaction: r =.019, P > 0.05) or with the relle-score (r = 0.094, P > 0.05). The relle-score was significantly associated with both outcome variables (neuroticism: r = 0.181, P < 0.005, life satisfaction: r = 0.223, P < 0.001). ANOVAs showed that 5-HTTLPR genotype groups did not differ with respect to any other variable, i.e. age (F 2,354 = 1.318), neuroticism (F 2,354 = 0.902), life satisfaction (F 2,354 = 1.942) and relle-score (F 2,354 =.788) (all P > 0.05). Men and women did not differ with respect to rellescores (F 1,355 = 1.610, P > 0.05) or life satisfaction scores (F 1,355 = 1.048, P > 0.05), however, men were slightly older ( ± 2.99 years) than women ( ± 2.73 years) (F 1,355 = , P < 0.001). Not surprisingly, men also reported significantly lower neuroticism scores (5.07 ± 3.21) than women (6.74 ± 3.15) (F 1,355 = , P < 0.001). A chi-square analysis showed that men and women did not differ with respect to genotype frequencies (χ 2 = 3.283, P > 0.05). Does 5-HTTLPR genotype moderate the association between the relative number of positive and negative LEs and the outcome variables (neuroticism and life satisfaction)? Our first hypothesis was to find an increased reactivity to environmental stimuli, regardless of their valence, in S-allele carriers. In other words, we expected the 5-HTTLPR genotype to moderate the effect of LEs on neuroticism or life satisfaction, respectively, in the SS- and LS-groups, but not in the LL-group. In order to test this assumption, we used hierarchical regression models, entering variables in the following order: age, sex (1: men, 2: women), relle-score, 5-HTTLPR genotype (0: LaLa-Grp, 1: LS-Grp, 2: SS-Grp) and interaction term relle-score 5-HTTLRP genotype. Figure 1: relle scores and life satisfaction association by genotype. Regression lines depicting the association between relle scores and life satisfaction, separated by genotype group. Regarding life satisfaction, only the interaction term significantly predicted life satisfaction scores (β = 0.287, P < 0.001; R 2 = 0.082; F 1,355 = , P < 0.001). With respect to neuroticism, sex as well as the interaction term were significant predictors (sex: β = 0.224, P < 0.001; interaction term β = 0.226, P < 0.001; model: R 2 = 0.10; F 1,354 = , P < 0.001). In order to test whether the moderation of the rele-score neuroticism association by the 5-HTTLPR genotype was different in men and women, we performed an additional hierarchical regression analysis entering the following variables: sex (1: men, 2: women), relle-score, 5-HTTLPR genotype (0: LaLa-Grp, 1: LS-Grp, 2: SS-Grp), the interaction term relle-score 5-HTTLRP genotype and the three-way interaction term relle-score 5-HTTLPR genotype sex. Indeed, the three-way interaction term as well as sex became significant (sex: β = 0.219, P < 0.001; three-way interaction term: β = 0.229, P < 0.001; model: R 2 = 0.10; F 1,354 = , P < 0.001). Post hoc tests using simple regression models separated by 5-HTTLPR genotype groups showed the strongest effect of relle-scores on life satisfaction in the SS-group. This association also was detected in the LS-group, albeit weaker. Within the LL-group, no association was detected (see Fig. 1). With respect to neuroticism scores, similar results were obtained in women. In men, however, we found an association between relle-scores and neuroticism within the SS-group only (see Fig. 2). Do S-allele carriers, especially SS-homozygotes, benefit more from a preponderance of positive LEs than LL-homozygotes? Our second study question concerned the hypothesis that S-allele carriers, especially SS-homozygotes, would benefit from a preponderance of positive LEs more than LLhomozygotes while also being damaged to a higher degree from a preponderance of negative LEs. Genes, Brain and Behavior (2012) 11:

4 Kuepperetal. (a) (b) Figure 3: Gene environment interaction on life satisfaction. Interaction effects between 5-HTTLPR genotype and LE groups with respect to life satisfaction. Mean scores and standard deviations (SD) of life satisfaction are given as a function of central 5-HTTLPR genotype groups and LE groups. Figure 2: relle scores and neuroticism association by genotype and sex. Regression lines depicting the association between relle scores and neuroticism, separated by genotype groups and sex. (a) men, (b) women. In order to test this assumption, we used an extreme group approach, only looking at subjects with a clear preponderance of positive or negative LEs; subjects with a relle-score of > = 3 were included in the preponderance of positive LEs group (n = 93). Those with relle scores of < = 3 were included in the preponderance of negative LEs group (n = 111). ANOVAs with two between-subject factors [factor 1: genotype, three levels (1: SS, 2: LS, 3: LL), factor 2: LEs, 2 levels (1: preponderance of positive LEs, 2: preponderance of negative LEs)] and life satisfaction as dependent variable revealed a main effect of LE groups (F 2,198 = , P < 0.001, η 2 = 0.043) as well as a significant genotype LE-group interaction effect (F 2,198 = 5.593, P < 0.005, η 2 = 0.037) (see Fig. 4). Post hoc group comparisons showed that SS-homozygotes, having experienced predominantly negative LEs, showed significantly reduced life satisfaction scores as compared to LL-homozygotes (F 2,198 = 7.752, P < 0.01) with the heterozygotes falling in between these groups. However, SS-subjects, having experienced predominantly positive LEs, had significantly increased life satisfaction scores as compared to LL-homozygotes (F 2,198 = 4.057, P < 0.05) with the heterozygote group again scoring in between these groups (also see Fig. 3). With respect to neuroticism, we again found a main effect of LE groups (F 2,197 = 8.142, P < 0.01, η 2 = 0.040) as well as a significant interaction effect between 5-HTTLPRgenotype group LEs groups (F 2,179 = 4.201, P > 0.05, η 2 = 0.041) not surprisingly sex, which was used as a covariant, also showed a significant effect (F 2,179 = 4.993, P < 0.05, η 2 =.025). Therefore, post hoc analyses were performed separated by sex (see Fig. 4). Within women, we found that SS-homozygotes who experienced predominantly positive LEs showed significantly reduced neuroticism scores compared to LL-homozygotes (F 1,34 = 4.764, P < 0.05) with the heterozygotes scoring in between groups. In men, we also found a non-significant tendency for the combination of SS-genotype with predominantly positive LEs to be associated with reduced neuroticism scores (F 1,17 = 2.092, P = 0.17]. Discussion We were interested in further testing the idea that S- allele carriers of the 5-HTTLPR were overall more strongly influenced by environmental stimuli, regardless of their valence, therefore the S-allele would constitute a genetic plasticity factor rather than a vulnerability factor. Specifically, the aims of our study were twofold: (1) do S-allele carriers, especially SS-homozygotes, show an increased reactivity to positive and negative LEs throughout their life spans? and (2) do S-allele carriers, especially SS-homozygotes, actually benefit more from a preponderance of positive LEs more than LL-homozygotes? With respect to our first question, we found that life satisfaction scores were associated with the relative number 646 Genes, Brain and Behavior (2012) 11:

5 5-HTTLPR S-allele as a genetic plasticity factor (a) (b) Figure 4: Gene environment interaction on neuroticism. Interaction effects between 5-HTTLPR genotype and LE groups with respect to neuroticism, separated by sex. (a) men, (b) women. Mean scores and standard deviations (SDs) of neuroticism are given as a function of central 5-HTTLPR genotype groups and LE groups. of positive and negative LEs throughout the life span in S- allele carriers. We found quite similar associations in women for neuroticism. In men, however, this association was only detected in SS-homozygotes. Within the LL-homozygotes, no association between LEs and personality was detected. So, we might conclude that the S-allele indeed appears to be associated with an increased reactivity to environmental stimuli regardless of their valence. These results are in accordance with data reported by Pluess et al. (2010) and Taylor et. al. (2006), to the knowledge of the authors the only other studies to date, systematically taking positive LEs into account when studying the interaction of 5-HTTLPR genotype and environmental stimuli as well as its associations with personality. As mentioned before, both studies showed an association of the relative number of positive and negative LEs experienced within the last 6 months and neuroticism scores within SShomozygotes only, with a preponderance of positive LEs being associated with reduced neuroticism scores. This effect was not found within the other two genotype groups. Here, our results are somewhat contradicting, since we found this association also in SL-heterozygotes, however, only in the female subsample. Several reasons for this discrepancy come to mind. For one, this might be a simple matter of statistical power, since both samples (Taylor: n = 118, Pluess: n = 118) were considerably smaller than ours (n = 357). This discrepancy might, however, also be due to the fact that we assessed LEs throughout the entire life span rather than only within the last 6 months. One might speculate that the impact of the accumulated LEs over the whole life span is somewhat stronger than of what has happened to a given person during a relatively short and recent period, thereby creating stronger associations. Future studies should look into this question in more detail. A third possible explanation is the effect of sex on the association between relative number of LEs and neuroticism scores as found in our sample. This effect was not detected within either of the other samples. Again, this might be due to smaller sample sizes, in which case the samples simply were too small to detect a possible effect of sex. Our male subsample is, however, still too small to conclusively interpret our data, and a replication would be needed in order to test whether men and women indeed systematically differ with respect to the effects of being S-allele carriers. It should be kept in mind, however, that several other studies do find sex-dependent associations between the S-allele and a variety of outcome variables (Brummett et al. 2008a,b; Du et al. 2000; Grabe et al. 2005; Mizuno et al. 2006; Sjoberg et al. 2006; Stoltenberg et al. 2010). Taken together, these studies might tentatively indicate that sex does impact the association between 5-HTTLPR and any given outcome regardless of other interacting predictors, such as LEs and that these effects should be analyzed in more detail. It would be feasible to assume that a variety of neuroendocrine factors which are sex-dimorphic and have been implemented in the regulation of emotional reactivity and personality might act as modulators of the discussed associations. Potential candidates might be the sexual steroids, e.g. testosterone and estrogen or peptide hormones such as oxytocin. In this context, the sexual steroids might be of special interest since several animal studies were able to show the 5-HT-system to interact with the sexual steroids (Dominguez et al. 2003; Fink et al. 1999; Flugge et al. 1998; Kindlundh et al. 2003; Sundblad et al. 1997). Interestingly, the effect of sex in our data was only found for neuroticism, but not life satisfaction. Even though both measures are highly correlated (r = 0.65), as would be expected, and appear to share substantial variance, they obviously are not interchangeable. Also in line with earlier studies, no sex-difference had been found with respect to life satisfaction. Furthermore, regardless of sex, life satisfaction was associated with the relative number of positive and negative LEs in SS-homozygotes and SL-heterozygotes but not in LL-homozygotes. This again indicates that the S-allele of the 5-HTTLPR is not per se a genetic risk factor but might be looked upon as a potential beneficial genetic constitution in particularly positive environments. This idea was tested further within our second study question: do S-allele carriers actually benefit from positive LEs more than LL-homozygotes? We found that, indeed the SS-homozygotes who experienced predomininantly positive LEs showed the highest life satisfaction scores as well as Genes, Brain and Behavior (2012) 11:

6 Kuepperetal. the lowest neuroticism scores. So, we might conclude that at least SS-homozygotes do benefit substantially from a preponderance of positive LEs, which was not found for LL-homozygotes, thereby further corroborating the plasticity hypothesis. It is particularly noteworthy that our data indicates that positive LEs might act as a kind of buffer for the adverse effects of negative LEs in susceptible genotype groups (SS and to a lesser degree SL). That is, even if a person with a susceptible 5-HTTLPR genotype has experienced a number of negative LEs, this person appears still to end up scoring low in neuroticism and high in life satisfaction if these negative events are counter-balanced by a sufficiently high number of positive LEs. These results might also, at least partially, explain why several studies were not able to replicate the 5-HTTLPR stressful LEs interaction on measures such as neuroticism or incidences of affective disorders in which positive LEs were not assessed (Middeldorp et al. 2010; Risch et al. 2009). Certain limitations of our data should be kept in mind when interpreting the results. First of all, it would be of great interest to conduct longitudinal designs in order to be able to make a more confident statement with respect to the causality of the effects described based on correlational data. Furthermore, even though our sample size is comparably large, the male subsample does not allow for conclusive interpretation of our data especially with regard to our second study question. Future studies should, therefore, aim at including (1) larger numbers of subjects and (2) equal numbers of men and women in order to verify whether the effect of sex we found is reproducible or rather a characteristic of the specific sample at hand. Another point to keep in mind is that we did not adjust for genetically inferred ethnicity. In order to avoid population stratification, it is sensible to either restrict one s study population to one ethnic group or to sample enough subjects to test several ethnic groups independently. However, due to the population structure in Germany, which is made up primarily of Caucasians, we chose to restrict our analysis to Caucasian subjects. Assessment of ethnicity was done by means of observer ratings. These ratings might be somewhat flawed, that is a certain proportion of ratings might be disconcordant with genetically inferred ethenicity (Chanock et al. 2007; Dumitrescu et al. 2010). However, observer ratings do appear to be very reliant, as some studies place the concordance between observer ratings and genetically referred ethnicity at around 98.5% (Dumitrescu et al. 2010). Therefore, effects on our results probably are small. A further limitation concerns the LEs assessed. Even though we aimed at including a large and representative scope of positive and negative LEs, our list is by no means exhaustive. Albeit that subjects were given the opportunity to list further events they had experienced throughout their lives and considered to have an impact on them, this does not guarantee that any given subject would recall all LEs without a primer. Therefore, some subjects might have experienced a given event but either deemed it not relevant enough to list or forgot to list it when filling in the questionnaire, while other subjects were more thorough when filling in the questionnaire. It might be interesting to expand the LEs assessed in future studies either by including items from further LEs questionnaires or by doing an exploratory study having a sufficiently large number of people from different backgrounds list all positive and negative experiences they considered relevant/important in their lives including any additional events in an expanded questionnaire. An important question mostly left unanswered until now is: how does it all work? Which mechanisms are responsible for the apparent increased environmental reactivity of S-allele carriers? Pluess et al. (2010) already pointed out that several studies show an overall increased neural reactivity of S-allele carriers at least to negative stimuli (Pluess et al. 2010). Furthermore, several studies showed S-allele carriers to exhibit an increased attentional bias for negative stimuli [for summary, see e.g. (Beevers et al. 2009)]. Fewer data are available regarding the question whether this holds true for positive stimuli as well. Several studies, however, showed an increased attentional bias for positive and negative stimuli in S-allele carriers (Beevers et al. 2009, 2011; Fox et al. 2011). Additionally, future studies should look at epigenetic effects in order to be able to close the gap between genotype, environmental stimuli and their isolated or combined impact on neural or attentional reactivity or personality, respectively. In conclusion, we believe that our data do support the idea that the S-allele of the 5-HTTLPR actually constitutes a genetic plasticity factor rather than a vulnerability factor, thereby rendering S-allele carriers more reactive to environmental stimuli, be they positive or negative in nature. We believe that these findings are a promising add-on to the often confirmed classical vulnerability hypothesis. References Alexander, N., Kuepper, Y., Schmitz, A., Osinsky, R., Kozyra, E. & Hennig, J. (2009) Gene-environment interactions predict cortisol responses after acute stress: implications for the etiology of depression. Psychoneuroendocrinology 34, Beevers, C.G., Marti, C.N., Lee, H.J., Stote, D.L., Ferrell, R.E., Hariri, A.R. & Telch, M.J. (2011) Associations between serotonin transporter gene promoter region (5-HTTLPR) polymorphism and gaze bias for emotional information. J Abnorm Psychol 120, Beevers, C.G., Wells, T.T., Ellis, A.J. & McGeary, J.E. (2009) Association of the serotonin transporter gene promoter region (5- HTTLPR) polymorphism with biased attention for emotional stimuli. J Abnorm Psychol 118, Belli, R.F., Shay, W.L. & Stafford, F.P. (2001) Event history calendars and question list surveys: a direct comparison of interviewing methods. Public Opin Q 65, Belsky, J., Jonassaint, C., Pluess, M., Stanton, M., Brummett, B. & Williams, R. (2009) Vulnerability genes or plasticity genes? Mol Psychiatry 14, Brummett, B.H., Boyle, S.H., Siegler, I.C., Kuhn, C.M., Ashley-Koch, A., Jonassaint, C.R., Zuchner, S., Collins, A. & Williams, R.B. (2008a) Effects of environmental stress and gender on associations among symptoms of depression and the serotonin transporter gene linked polymorphic region (5-HTTLPR). Behav Genet 38, Brummett, B.H., Muller, C.L., Collins, A.L., Boyle, S.H., Kuhn, C.M., Siegler, I.C., Williams, R.B. & Ashley-Koch, A. (2008b) 5-HTTLPR 648 Genes, Brain and Behavior (2012) 11:

7 5-HTTLPR S-allele as a genetic plasticity factor and gender moderate changes in negative affect responses to tryptophan infusion. Behav Genet 38, Caspi, A., Hariri, A.R., Holmes, A., Uher, R. & Moffitt, T.E. (2010) Genetic sensitivity to the environment: the case of the serotonin transporter gene and its implications for studying complex diseases and traits. Am J Psychiatry 167, Caspi, A., Sugden, K., Moffitt, T.E., Taylor, A., Craig, I.W., Harrington, H., McClay, J., Mill, J., Martin, J., Braithwaite, A. & Poulton, R. (2003) Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 301, Chanock, S.J., Manolio, T., Boehnke, M. et al. (2007) Replicating genotype-phenotype associations. Nature 447, Dominguez, R., Cruz-Morales, S.E., Carvalho, M.C., Xavier, M. & Brandao, M.L. (2003) Effect of steroid injection to newborn rats on serotonin activity in frontal cortex and raphe. Neuroreport 14, Du, L., Bakish, D. & Hrdina, P.D. (2000) Gender differences in association between serotonin transporter gene polymorphism and personality traits. Psychiatr Genet 10, Dumitrescu, L., Ritchie, M.D., Brown-Gentry, K., Pulley, J.M., Basford, M., Denny, J.C., Oksenberg, J.R., Roden, D.M., Haines, J.L. & Crawford, D.C. (2010) Assessing the accuracy of observerreported ancestry in a biorepository linked to electronic medical records. Genet Med 12, Eley, T.C., Sugden, K., Corsico, A., Gregory, A.M., Sham, P., McGuffin, P., Plomin, R. & Craig, I.W. (2004) Gene-environment interaction analysis of serotonin system markers with adolescent depression. Mol Psychiatry 9, Fahrenberg, J., Hampel, R. & Selg, H. (1984) Das Freiburger Persönlichkeitsinventar (FPI-R). Fink, G., Sumner, B., Rosie, R., Wilson, H. & McQueen, J. (1999) Androgen actions on central serotonin neurotransmission: relevance for mood, mental state and memory. Behav Brain Res 105, Flugge, G., Kramer, M., Rensing, S. & Fuchs, E. (1998) 5HT1Areceptors and behaviour under chronic stress: selective counteraction by testosterone. Eur J Neurosci 10, Fox, E., Zougkou, K., Ridgewell, A. & Garner, K. (2011) The serotonin transporter gene alters sensitivity to attention bias modification: evidence for a plasticity gene. Biol Psychiatry 70, Grabe, H.J., Lange, M., Wolff, B., Volzke, H., Lucht, M., Freyberger, H.J., John, U. & Cascorbi, I. (2005) Mental and physical distress is modulated by a polymorphism in the 5-HT transporter gene interacting with social stressors and chronic disease burden. Mol Psychiatry 10, Gray, M.J., Litz, B.T., Hsu, J.L. & Lombardo, T.W. (2004) Psychometric properties of the life events checklist. Assessment 11, Holmes, T.H. & Rahe, R.H. (1967) The Social Readjustment Rating Scale. J Psychosom Res 11, Kindlundh, A.M., Lindblom, J., Bergstrom, L. & Nyberg, F. (2003) The anabolic-androgenic steroid nandrolone induces alterations in the density of serotonergic 5HT1B and 5HT2 receptors in the male rat brain. Neuroscience 119, Lesch, K.P., Bengel, D., Heils, A., Sabol, S.Z., Greenberg, B.D., Petri, S., Benjamin, J., Muller, C.R., Hamer, D.H. & Murphy, D.L. (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, Middeldorp, C.M., de Geus, E.J., Willemsen, G., Hottenga, J.J., Slagboom, P.E. & Boomsma, D.I. (2010) The serotonin transporter gene length polymorphism (5-HTTLPR) and life events: no evidence for an interaction effect on neuroticism and anxious depressive symptoms. Twin Res Hum Genet 13, Mizuno, T., Aoki, M., Shimada, Y., Inoue, M., Nakaya, K., Takahashi, T., Itoyama, Y., Kanazawa, M., Utsumi, A., Endo, Y., Nomura, T., Hiratsuka, M., Mizugaki, M., Goto, J., Hongo, M. & Fukudo, S. (2006) Gender difference in association between polymorphism of serotonin transporter gene regulatory region and anxiety. J Psychosom Res 60, Munafo, M.R., Johnstone, E.C., Wileyto, E.P., Shields, P.G., Elliot, K.M. & Lerman, C. (2006) Lack of association of 5-HTTLPR genotype with smoking cessation in a nicotine replacement therapy randomized trial. Cancer Epidemiol Biomarkers Prev 15, Munafo, M.R., Brown, S.M. & Hariri, A.R. (2008) Serotonin transporter (5-HTTLPR) genotype and amygdala activation: a metaanalysis. Biol Psychiatry 63, Munafo, M.R., Freimer, N.B., Ng, W., Ophoff, R., Veijola, J., Miettunen, J., Jarvelin, M.R., Taanila, A. & Flint, J. (2009) 5-HTTLPR genotype and anxiety-related personality traits: a meta-analysis and new data. Am J Med Genet B Neuropsychiatr Genet 150B, Nakamura, M., Ueno, S., Sano, A. & Tanabe, H. (2000) The human serotonin transporter gene linked polymorphism (5-HTTLPR) shows ten novel allelic variants. Mol Psychiatry 5, Pluess, M., Belsky, J., Way, B.M. & Taylor, S.E. (2010) 5-HTTLPR moderates effects of current life events on neuroticism: differential susceptibility to environmental influences. Prog Neuropsychopharmacol Biol Psychiatry 34, Risch, N., Herrell, R., Lehner, T., Liang, K.Y., Eaves, L., Hoh, J., Griem, A., Kovacs, M., Ott, J. & Merikangas, K.R. (2009) Interaction between the serotonin transporter gene (5-HTTLPR), stressful life events, and risk of depression: a meta-analysis. JAMA 301, Sarason, I.G., Johnson, J.H. & Siegel, J.M. (1978) Assessing the impact of life changes development of the life experiences survey. J Consult Clin Tsychol 46, Schinka, J.A., Busch, R.M. & Robichaux-Keene, N. (2004) A metaanalysis of the association between the serotonin transporter gene polymorphism (5-HTTLPR) and trait anxiety. Mol Psychiatry 9, Sen, S., Burmeister, M. & Ghosh, D. (2004) Meta-analysis of the association between a serotonin transporter promoter polymorphism (5-HTTLPR) and anxiety-related personality traits. Am J Med Genet B Neuropsychiatr Genet 127B, Sjoberg, R.L., Nilsson, K.W., Nordquist, N., Ohrvik, J., Leppert, J., Lindstrom, L. & Oreland, L. (2006) Development of depression: sex and the interaction between environment and a promoter polymorphism of the serotonin transporter gene. Int J Neuropsychopharmacol 9, Stoltenberg, S.F. & Vandever, J.M. (2010) Gender moderates the association between 5-HTTLPR and decision-making under ambiguity but not under risk. Neuropharmacology 58, Sundblad, C. & Eriksson, E. (1997) Reduced extracellular levels of serotonin in the amygdala of androgenized female rats. Eur Neuropsychopharmacol 7, Taylor, S.E., Way, B.M., Welch, W.T., Hilmert, C.J., Lehman, B.J. & Eisenberger, N.I. (2006) Early family environment, current adversity, the serotonin transporter promoter polymorphism, and depressive symptomatology. Biol Psychiatry 60, Wendland, J.R., Martin, B.J., Kruse, M.R., Lesch, K.P. & Murphy, D.L. (2006) Simultaneous genotyping of four functional loci of human SLC6A4, with a reappraisal of 5-HTTLPR and rs Mol Psychiatry 11, Willis-Owen, S.A., Turri, M.G., Munafo, M.R., Surtees, P.G., Wainwright, N.W., Brixey, R.D. & Flint, J. (2005) The serotonin transporter length polymorphism, neuroticism, and depression: a comprehensive assessment of association. Biol Psychiatry 58, Acknowledgments This study has been supported by the German Research Foundation (DFG, HE ). None of the authors have conflicting interests to report. Genes, Brain and Behavior (2012) 11:

8 Kuepperetal. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1: Life events assessed. For each life event, the average number of occurrences and standard deviations are given as well as the total number of reported occurrences within the whole sample. Furthermore, the average valence ratings plus standard deviations are given. Following Sarrason et al. (1978), events were classified as positive or negative depending on the individual ratings of the subjects. Valence ratings were scored as follows: 1, very positive; 2, positive; 3, neutral; 4, negative; 5, very negative. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. 650 Genes, Brain and Behavior (2012) 11: