Interaction between genetic polymorphisms and stressful life events in first episode depression

Journal of Affective Disorders 119 (2009) 107–115

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Journal of Affective Disorders j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j a d

Research report

Interaction between genetic polymorphisms and stressful life events in first episode depression Jens Drachmann Bukh a,⁎, Camilla Bock a, Maj Vinberg a, Thomas Werge b, Ulrik Gether c, Lars Vedel Kessing a a b c

Department of Psychiatry, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark Research Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenhagen, Denmark Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Denmark

a r t i c l e

i n f o

Article history: Received 6 January 2009 Received in revised form 26 February 2009 Accepted 26 February 2009 Available online 31 March 2009 Keywords: Depressive disorder Stressful life events Genetic polymorphism Gene–environment interaction Serotonin transporter Brain derived neurotrophic factor

a b s t r a c t Background: A polymorphism in the serotonin transporter (5-HTT) gene seems to moderate the influence of stressful life events on depression. However, the results from previous studies of gene–environment interactions in depression are inconsistent and might be confounded by the history of depression among participants. Method: We applied a case-only design, including 290 ethnically homogeneous patients suffering exclusively from first episode depression. Psychiatric mo-morbidity, personality traits and disorders and stressful life events in a six months period preceding onset of depression were evaluated by means of interviews and questionnaires. Additionally, we genotyped nine polymorphisms in the genes encoding the serotonin transporter, brain derived neurotrophic factor, catechol-O-methyltransferase, angiotensin converting enzyme, tryptophane hydroxylase, and the serotonin receptors 1A, 2A, and 2C. Results: The low activity variants of the 5-HTT-linked polymorphic region in the serotonin transporter gene and the Met-allele of a single nucleotide polymorphism (Val66Met) in the gene encoding brain derived neurotrophic factor were independently associated with the presence of stressful life events prior to onset of depression, also when corrected for the effect of age, gender, marital status, personality disorder, neuroticism, and severity of depressive symptoms at the time of interview. Conclusion: Polymorphisms in the genes encoding the serotonin transporter and the brain derived neurotrophic factor interact with recent stressful life events on depression among patients with no history of previous depressive episodes. © 2009 Elsevier B.V. All rights reserved.

1. Introduction In 2003, Caspi et al. (2003) demonstrated that an insertion/ deletion polymorphism in the serotonin transporter gene, 5-HTT-linked polymorphic region (5-HTTLPR), moderated the influence of stressful life events (SLE) on depression. Carriers

⁎ Corresponding author. Department of Psychiatry, University Hospital of Copenhagen, Rigshospitalet, Blegdamsvej 9, DK 2100 Copenhagen, Denmark. Tel.: +45 3545 6230; fax: +45 3545 6218. E-mail addresses: [email protected], [email protected] (J. Drachmann Bukh). 0165-0327/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jad.2009.02.023

of the short low-expression allele (S) were more likely to develop depression following exposure to SLE or childhood adversities compared to individuals homozygous for the long high-expression allele (L). A number of studies have replicated the association between the S-allele and vulnerability to depression, however, as discussed below, the results to date are not entirely consistent (Uher and McGuffin, 2008). Subsequently, a range of single nucleotide polymorphisms (SNPs) have been identified in the serotonin transporter gene, among these an A/G substitution within the 5-HTTLPR (Nakamura et al., 2000). Only the long/A allele (LA) seems to result in higher expression of the gene, whereas the long/G

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allele (LG) is more similar to the S-allele with lower levels of mRNA and low receptor density (Praschak-Rieder et al., 2007). Four of the previous studies (Zalsman et al., 2006; Chorbov et al., 2007; Kilpatrick et al., 2007; Power et al., 2008) have taken into account this SNP and grouped the alleles in low activity (S + LG) and high activity (LA) variants. Besides the 5-HTTLPR, SNPs in the genes encoding brain derived neurotrophic factor (BDNF) (Kaufman et al., 2006; Kim et al., 2007; Wichers et al., 2008) and catechol-O-methyltransferase (COMT) (Mandelli et al., 2007) have been associated with vulnerability to depression following SLE. The genetic makeup is likely to vary between patients with fewer depressive episodes compared to patients with many previous episodes. For example, Zalsman et al. (2006) found that the low activity alleles of 5-HTTLPR predicted more lifetime depressive episodes (Fig. 1, pathway c). Further, it is well established that the prevalence of SLE is high among patients with first episode depression or only a few episodes (60–70%) and lower among patients who have experienced many previous depressive episodes (20–40%) (Post, 1992; Kendler et al., 2000). Thus, the association between SLE and depression declines with increasing number of episodes (Fig. 1, pathway b). Additionally, this proposed change in reactivity to stressors across the course of repeated episodes may itself be modified by genetic factors (Kendler et al., 2001) (Fig. 1, pathway d). Nevertheless, only few studies on genetic modifiers of the stress–depression relationship have distinguished between first onset and recurrence of depression or otherwise accounted for the confounding effect of the history of depression (Caspi et al., 2003; Gillespie et al., 2005; Wilhelm et al., 2006; Mandelli et al., 2007). Consequently, the variation in the findings between studies might be a consequence of different samples of patients with various numbers of previous depressive episodes. The present study aimed to evaluate the interaction between SLE and genetic polymorphisms in a sample of patients suffering exclusively from first episode depression, thereby eliminating the confounding effect of the history of depression. Patients were consecutively sampled from the Danish Psychiatric Central Research Register. 2. Methods 2.1. Design We applied a case-only design, which is an approach used to evaluate gene–environment interaction in disease aetiol-

ogy (Khoury and Flanders, 1996). In case-only studies, the association between an exposure (in this study SLE) and the genotype is examined among case subjects only (in this study subjects with first episode depression). The case selection follows the principles in a traditional case–control study, but the exposure effect is assessed only among cases. The cases with and without the susceptibility genotype are compared with respect to the environmental risk factor, and odds ratios with confidence intervals are obtained using standard analyses including multivariate models to adjust for other potentially confounding variables. In a traditional case–control design, odds ratios for the effects of the exposure alone (ORe) and the genotype alone (ORg) can be compared with joint odds ratios for the environmental exposure and the genotype (ORge). A synergy index (ORge/ORexORg) above one indicates more than multiplicative effects between the exposure and the genotype, while a synergy index less than one indicates less then multiplicative effects. With independence assumed between the exposure and the genotype, it can be shown that the odds ratio in a case-only study relating the exposure and the genotype equals the synergy index on a multiplicative scale derived from a regular case–control study. Consequently, an odds ratio significantly greater than one obtained in a caseonly study indicates an interacting effect between the allelic variants and the environmental exposure, even though the main effects of the genetic and the environmental factors cannot be estimated (Khoury and Flanders, 1996). The method has previously been used by Mandelli et al. to study interactions between SLE and various polymorphisms (Mandelli et al., 2007). 2.2. The register The Danish Psychiatric Central Research Register is a nation-wide registration of all psychiatric hospitalisations and outpatient contacts (patients in ambulatory care or community psychiatry centres) in Denmark (Munk-Jorgensen and Mortensen, 1997). The register comprises information on treatment settings, duration of contact to psychiatric care, and psychiatric diagnoses (from 1994 January 1 according to The International Classification of Diseases, 10th Revision, ICD-10 (World Health Organisation, 1992)). The registration is based on a unique person identification number assigned to all inhabitants in Denmark (Civil Person Registration number), thus previous contacts to psychiatric services can be established with great certainty irrespective of changes in names or

Fig. 1. Relations between stressful life events (SLE), depression, genes, and the course of illness. (a) Interaction between genes and SLE on the risk of developing a depressive episode (gene–environment interaction). (b) Interaction between SLE and course of illness on the risk of developing a new depressive episode (sensitisation). (c) Genetic influence on the course of illness. (d) Three-way interaction between SLE, genes and course of illness on the risk of developing a new depressive episode. (e) Genetic influence on the risk of experiencing SLE (gene–environment correlation).

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addresses. General practitioners and psychiatrists in private practice do not report to the register. No private psychiatric hospitals are operating in Denmark. 2.3. The sample The study sample was defined as all in- and outpatients with the diagnosis of a single depressive episode (ICD-10, code DF32–32.9) reported to the Danish Psychiatric Central Research Register following the first contact ever to a psychiatric hospital in the astern part of Denmark (Zealand (Sjælland)). This area comprises approximately 2.4 million inhabitants corresponding to 44% of the total Danish population. Patients were sampled consecutively from the register every second month in a period from 2005 June 1 through 2007 May 31 and invited to participate in the study 1– 3 months after discharge. In order to obtain a homogeneous sample, only ethnic Danes aged 18–70 at the time of discharge were included in the study. Danish ethnicity was established when the proband as well as both parents were born in Denmark and no grandparents were born outside Europe. As we also intended to investigate medical treatment outcome, patients were included only if the current episode was treated with antidepressant medication in one week or longer. The exclusion criteria were significant physical illness, dementia or mental retardation. The Danish Ministry of Health, The Danish Ethic Committee (KF 01.209/04) and the Data Inspection approved the study. The investigation was conducted in accordance with the latest version of the Declaration of Helsinki. All participants gave written informed consent. 2.4. Interviews and questionnaires The interviews were conducted by two experienced medical doctors (CB and JDB) using standardized semistructured interviews. The interviewers conducted co-ratings of 10 interviews in a pilot study and of additional 16 interviews during the 2-year inclusion period. In order to validate the clinical diagnoses reported to the register, psychiatric diagnoses according to ICD-10 criteria were obtained using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) (Wing et al., 1990). The diagnostic evaluation was based on the interview with the patient and data from case reports, which were available for 79.1% of the participants. ICD-10 diagnoses according to the SCAN interview were established for the episode leading to psychiatric hospital contact (the month with most pronounced symptoms) and for the lifetime before. Both interviewers followed a WHO-certified course in the use of the SCAN. The reliability coefficient (agreement of the diagnosis of a single depressive episode) was 1.0. Diagnoses of personality disorders were assessed according to DSM-IV criteria using the Structured Clinical Interview for DSM-IV Axis II Personality Disorders (SCID-II) (First et al., 1997). The reliability coefficient (agreement of the diagnosis of one or more personality disorders) was 0.76. The presence of life events in a period of six months prior to onset of the depressive symptoms was evaluated by means of the Interview for Recent Life Events (IRLE) (Paykel, 1997), which specifies 64 different life events in nine areas com-

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prising work, education, financial conditions, health, bereavement, migration, courtship and cohabitation, legal matters, family and social affairs, and marital relations. For each life event, detailed information was recorded in order to assess the time of occurrence, the independency of the depressive state (rated from almost certainly independent to almost certainly dependent on a five point scale) and the negative impact of the event (rated from no negative impact to severe negative impact on a five point scale). In line with the guidelines for the use of IRLE, the evaluation of negative impact was based on an objective judgement made by the interviewer taking into account the individual and contextual circumstances. Stressful life events were defined as those events, which were rated certainly or most likely independent of the depression (an independency score of 1–2) and of moderate to severe negative impact (an impact score of 3–5). The reliability coefficient (agreement of the presence of one or more stressful life events) was 0.82. Severity of depressive symptoms at the time of the interview was assessed using the 17-item Hamilton Depression Rating Scale (HDRS) (Hamilton, 1960). In addition, the participants completed the 21-item Beck Depression Inventory (BDI) (Beck et al., 1961), and the Eysenck Personality Questionnaire (EPQ) in order to calculate the neuroticism (n-scale) score. 2.5. Non-participants Register information on age, gender, treatment settings (inpatient/outpatient), diagnoses, and the duration of psychiatric hospital contact were available for all participants as well as non-participants in the study. Additional information was collected for non-participants on ethnicity and adverse life events prior to onset of depression by a structured telephone interview or in the alternative by means of a questionnaire including the same questions, which was posted to all non-participants. 2.6. Genotyping Nine polymorphisms in genes previously associated with depression and/or susceptibility to stressors were chosen for genotyping. DNA was isolated from full-blood using standard procedures. Genotyping of all SNPs and length polymorphisms were performed by allelic discrimination either using the Taqman technology or restriction fragment length polymorphism (PCR-RFLP) as follows: SERT (Serotonin Transporter, SLC6A4, Solute carrier family 6, member 4; Chr17): The length polymorphism (5-HTTLPR long/short) and the SNP (rs25531) in the promoter region of SERT were genotyped as described by Rasmussen and Werge (2007). COMT (cathechol-O-methyltransferase; Chr22): rs4680 (A/G) was genotyped using a commercially available ABI Taqman assay (Assay ID: C__25746809_50). BDNF (Brain-derived neurotrophic factor; Chr11): rs6265 (C/T) was genotyped using a commercially available ABI Taqman assay (Assay ID: C__11592758_10). ACE (angiotensin I converting enzyme [peptidyl-peptidase A]) 1; Chr17): rs4291 (A/T) was genotyped using a commercially available ABI TaqMan assays (Assay ID: C__11942507_10). TPH1 (tryptophan hydroxylase 1 (tryptophan 5-monooxygenase; Chr11): rs1800532 (C/A)

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was genotyped using a Tagman assay as described by Christiansen et al. (2007). HTR1A (5HT receptor 1A; Chr5): The C-1019G polymorphism was genotyped as described by Strobel et al. (2003). HTR2A (5HT receptor 2A; Chr13): The T102C polymorphism (rs6313) was genotyped as described by Bondy et al. (2000). HTR2C (5-hydroxytryptamine receptor 2C; ChrX): rs3813929 C/T was genotyped using a commercially available ABI Taqman assay (Assay ID: C__ 27488117_10). The commercially available ABI Taqman assays were delivered from Applied Biosystems Inc. and used according to the instructions provided by the supplier. 2.7. Statistical analysis Patients with different genotypes were compared with regard to demographic and clinical variables and the presence of one or more SLE preceding onset of depression using chisquare tests for categorical data and t-tests for continuous data. Significant associations between polymorphisms and exposure to SLE found in univariate analyses were further tested in logistic regression models analysing whether the polymorphism of interest predicted SLE (occurrence of one or more SLE vs. no SLE) when adjusted for the effect of age,

gender, marital status, comorbid personality disorder, neuroticism and HDRS score. The SS genotype of 5-HTTLPR was compared with SL and LL grouped together in accordance with Kendler et al. (2005) (below designated 5-HTTLPR (old genotypes)). Besides the long (L) and short (S) alleles of 5-HTTLPR, the alleles were grouped according to functionality (the high expression LA-allele vs. the low-expression S- and LG-alleles), and the genotype homozygous for the lowexpression alleles (S/S, S/LG, LG/LG) was compared with the genotypes comprising one or two high-expression alleles (S/ LA, LA/LG, and LA/LA) (below designated 5-HTTLPR (with A/ G)). The MetMet genotype of BDNF Val66Met was grouped together with ValMet and compared with ValVal in accordance with all previous studies of interaction between SLE and this polymorphism (Kaufman et al., 2006; Kim et al., 2007; Wichers et al., 2008). All other polymorphism were analysed in a full model containing the homozygous genotype as well as the two heterozygous genotypes, since there was not sufficient basis for a-priori assumption about recessive or dominant effects of any of these alleles. p-values (2-sided) b0.05 were regarded to indicate statistical significance in all analyses. The tests were performed with SPSS 15.0 for windows (Release 15.0.0 (6 Sep 2006)).

Table 1 Socio-demographic and clinical characteristics of the sample, totally and according to the presence or absence of stressful life events preceding onset of depression. Characteristic

Total (N = 290)

Gender (female), N (%) Age, median (quartiles) Education b, N (%) University degree Middle-range education High school Vocational education Elementary school Marital status c, N (%) Married/living together Living alone Family history of depression among first-degree relatives, N (%) Severity of depression d, N (%) Mild Moderate Severe Melancholic features, N (%) Psychiatric comorbidity, N (%) Anxiety/OCD Alcohol abuse Drug abuse Somatoform and eating disorders Personality disorders, N (%) Cluster A Cluster B Cluster C Depressive Any personality disorder Neuroticism-score e, mean (S.D.) Symptom severity at interview according to HDRS, mean (S.D.) Symptom severity at interview according to BDI f, mean (S.D.)

190 (65.5) 38.5 (28.3–52.4) 25 (8.7) 63 (21.9) 34 (11.8) 98 (34.0) 68 (23.6) 145 (54.5) 121 (45.4) 83 (28.6) 69 159 62 189

Stressful life event preceding onset Yes (N = 185)

No (N = 105)

111 (60.0) 43.9 (32.3–54.0)

79 (75.2) 33.1 (25.4–42.8)

19 43 21 66 34

(10.4) (23.5) (11.5) (36.1) (18.6)

97 (56.1) 76 (43.9) 54 (29.2)

6 20 13 32 34

pa

0.009 b 0.0005

(5.7) (19.0) (12.4) (30.5) (32.4)

0.1

48 (51.6) 45 (48.4) 29 (27.6)

0.5 0.8

(23.8) (54.8) (21.4) (65.2)

42 96 47 131

(22.7) (51.9) (25.4) (70.8)

27 63 15 58

(25.7) (60.0) (14.3) (55.2)

0.09

0.007

139 (47.9) 43 (14.8) 22 (7.6) 13 (4.5)

87 32 11 7

(47.0) (17.3) (5.9) (3.8)

52 11 11 6

(49.5) (10.5) (10.5) (5.7)

0.7 0.1 0.2 0.4

9 (3.1) 35 (12.1) 49 (16.9) 23 (7.9) 89 (30.7) 11.5 (6.3) 9.2 (6.2) 15.2 (10.0)

6 16 36 13 55 11.0 9.4 14.9

(3.2) (8.6) (19.5) (7.0) (29.7) (6.4) (6.5) (10.1)

3 19 13 10 34 12.4 8.9 15.6

(2.9) (18.1) (12.4) (9.5) (32.4) (6.1) (5.5) (9.7)

0.9 0.02 0.1 0.5 0.7 0.1 0.5 0.6

a p-values (2-sided) in analyses comparing patients who have experienced one or more SLE with those reporting no SLE (χ2-test for categorical data and t-test for continuous data). b N = 288. c N = 266. d According to the ICD-10 classification obtained by SCAN interview. e N = 235. f N = 274.

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3. Results 3.1. Participants and non-participants A total number 1486 patients with a main diagnosis of a single depressive episode were sampled from the register. Among the 1486 individuals, 480 were excluded (owing to data protection (N = 78), non-pharmacological treatment (N = 78), non-Danish ethnicity (N = 291), death (N = 9), disability (N = 14), or migration (N = 10)). Among the 1006 potential participants, 399 (39.7%) completed the full face-to-face interview. Besides a significant excess of women participating in the study (64.9% vs. 58.5%, p = 0.04), the participants did not differ from the nonparticipants with respect to age at discharge, severity of depression (mild, moderate or severe according to register diagnoses), setting (in- or outpatients), or duration of hospital contact (p N 0.1). Among the non-participants, 238 individuals (39.2%) completed the short questionnaire or telephone interview, and 184 of them (77.3%) reported one or more SLE before onset of depression. In comparison, 303 of the participants (75.9%) experienced one or more SLE (p = 0.7; the degree of independence was not included in this analysis since this aspect was not assessed in the telephone interviews). The diagnosis of a single depressive episode according to ICD-10 was established by the SCAN interview for 301 individuals (the remaining participants obtained diagnoses of recurrent depressive disorder (11.0%), schizophrenia (1.0%), bipolar disorder (3.3%), or other diagnoses (8.8%)). Blood samples were obtained from 290 individuals, who constituted the sample for further analyses in the present study. 3.2. Socio-demographic and clinical characteristics Totally, 63.9% of the patients with a single depressive episode had experienced one or more SLE within the six months preceding onset of depressive symptoms. Differences between patients with and without SLE are shown in Table 1. The gender distribution in both groups reflects the higher prevalence of depression among women, though the excess of female was significantly increased in the group reporting no SLE. Moreover, the patients with SLE were characterized by older age (median age 43.9 vs. 33.1, p b 0.0005), a higher prevalence of melancholic features (70.8% vs. 55.2%, p = 0.007) and less prevalent comorbid cluster B personality disorder (8.6% vs. 18.1%, p = 0.02). There was a trend for patients with SLE to have developed more often a severe depression according to the ICD-10 diagnoses (25.4% vs. 14.3%, p = 0.09). At the time of interview, there was no difference in the severity of depressive symptoms as measured on either HDRS or BDI. 3.3. Genotypes In the sample, none of the genetic polymorphisms departed significantly from Hardy–Weinberg equilibrium (5-HTTLPR (old genotypes): p = 0.5, 5-HTTLPR (with A/G): p = 0.2, BDNF: p = 0.1, COMT: p = 1.0, TPH1: p = 0.9, ACE: p = 0.7, 5-HTR1A: p = 1.0, 5-HTR2A: p = 0.4, and 5-HTR2C: p = 0.8). The allele frequencies resembled those described for Caucasians in HapMap (The International HapMap Project:

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www.hapmap.org/) and dbSNP (The Single Nucleotide Polymorphism database; www.ncbi.nlm.nih.gov/projects/SNP/). As can be seen from Table 2, SLE were significantly more frequent among patients with the low activity genotypes of 5-HTTLPR (with A/G) and the ValMet or MetMet genotypes of BDNF Val66Met, respectively. The prevalence of SLE among patients with the SS, SL and LL genotypes of 5-HTTLPR (old genotypes) were close to the results obtained when also considering the A/G substitution, although, using this categorization, the association with SLE only reached borderline significance (p = 0.07). The distribution of SLE according to the remaining polymorphisms did not show any significant associations. The effects of the 5-HTTLPR (with A/G) and BDNF Val66Met genotypes on the presence of SLE were further analysed in logistic regression models with the inclusion of gender, age, marital status, the diagnosis of a comorbid personality disorder (yes/no), and neuroticism score. In standard regressions with all variables included, the susceptibility genotypes

Table 2 Genotypes of 290 patients with first episode depression according to the presence or absence of stressful life events prior to onset (crude data). Genotype

Stressful life events preceding onset N (%) Yes (N = 185)

5-HTTLPR (with A/G) High activity Medium activity Low activity 5-HTTLPR (old genotypes) LL SL SS COMT b ValVal ValMet MetMet BDNF ValVal ValMet MetMet ACE AA AT TT TPH AA AC CC 5-HTR1A CC CG GG 5-HTR2A b CC CT TT 5-HTR2C CC CT TT

pa

No (N = 105)

50 (61.7) 80 (59.3) 55 (74.3)

31 (38.3) 55 (40.7) 19 (25.7)

0.03

60 (60.6) 84 (61.8) 41 (74.5)

39 (39.4) 52 (38.2) 14 (25.5)

0.07

36 (65.5) 89 (64.5) 60 (62.5)

19 (34.5) 49 (35.5) 36 (37.5)

0.9

101 (58.0) 69 (71.9) 15 (75.0)

73 (42.0) 27 (28.1) 5 (25.0)

0.01

74 (60.7) 92 (67.6) 19 (59.4)

48 (39.3) 44 (32.4) 13 (40.6)

26 (63.4) 90 (65.7) 69 (61.6)

15 (36.6) 47 (34.3) 43 (38.4)

0.8

46 (65.7) 90 (60.4) 49 (69.0)

24 (34.3) 59 (39.6) 22 (31.0)

0.4

31 (67.4) 86 (58.5) 67 (69.8)

15 (32.6) 61 (41.5) 29 (30.2)

0.2

136 (65.1) 32 (56.1) 17 (70.8)

73 (34.9) 25 (43.9) 7 (29.2)

0.3

0.4

a p-values (2-sided) in χ2-test comparing genotypes. 5-HTTLPR (with A/G) is categorized into high activity genotype vs. medium and low activity genotypes. 5-HTLLPR (old genotypes) is categorized into SS vs. SL and LL. BDNF is categorized into ValVal vs. ValMet and MetMet. b One missing genotype (N = 289).

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were both associated with approximately 2 times increased odds for having experienced one or more SLE prior to onset of depression (5-HTTLPR (with A/G): OR = 2.1, 95% CI 1.0–4.3, p = 0.05, BDNF Val66Met: OR = 1.8, 95% CI 1.0–3.4, p = 0.05). Following backward stepwise removal of insignificant variables, genotype and age was the only variables in both models, which contributed significantly to the presence of SLE (5-HTTLPR (with A/G): OR = 1.8, 95% CI 1.0–3.3, p = 0.05, BDNF Val66Met: OR = 2.0, 95% CI 1.2–3.4, p = 0.008). Since the univariate analyses had shown a significant association between SLE and cluster B personality disorder, but no associations with other, we repeated the analyses including cluster B personality disorder instead of the diagnosis of any personality disorder. In these models, age and genotypes were still the only variables significantly predicting the presence of SLE. Also, including the HDRS score at the time of interview in the models together with age and genotypes resulted in no substantial changes of odds ratios or confidence intervals. Finally, we considered, simultaneously, the effect of 5-HTTLPR (with A/G) and BDNF Val66Met and the covariates (age, gender, marital status, personality disorder and neuroticism) in a logistic regression analysis. In the standard regression model the effects of BDNF Val66Met and 5-HTTLPR (with A/G) on the presence of SLE were both significant (5-HTTLPR (with A/G): OR = 2.2, 95% CI 1.0–4.6, p = 0.04, BDNF Val66Met: OR = 2.2, 95% CI 1.0–3.6, p = 0.04), and again stepwise backward removal of variables resulted in a final model including the two genotypes and age (5-HTTLPR: OR = 2.0, 95% CI: 1.1–3.6, p = 0.03, BDNF Val66Met: OR = 2.1, 95% CI: 1.3–3.6, p = 0.005). Testing for interaction, the effect of 5-HTTLPR (with A/G) × BDNF Val66Met on SLE was not significant (p = 0.7). 4. Discussion 4.1. Main results We investigated the incidence of SLE within six months prior to onset of the first depressive episode and genotyped nine different genetic polymorphisms. We found significant associations between 5-HTLLPR (with A/G) and BDNF Val66Met, respectively, and exposure to one or more SLE. Both genotypes were independently associated with the presence of SLE, also when adjusted for the effects of age, gender, marital status, neuroticism and comorbid personality disorder. Since the positive findings were replications of prior results, we did not consider adjustment for multiple testing to be required. Yet, it should be noted that the effect of 5-HTTLPR did not withstand Bonferroni correction, and for that reason, the finding of an association between 5-HTTLPR and SLE might be regarded just as a trend. Finally, we found no effect of the interaction between 5-HTTLPR (with A/G) and BDNF Val66Met on exposure to SLE, and, further, we found no association between SLE and polymorphisms in COMT, TPH1, ACE, 5-HTR1A, 5-HTR2A, or 5-HTR2C, respectively. 4.2. Advantages First of all, we consider the inclusion of patients exclusively with first episode depression to be an important advan-

tage. Moreover, the study participants were systematically recruited, ethnically homogenous, and with a wide age range. Besides register data, we have additional information on about 40% of all non-participants. Based on these data, our sample seems to be representative for Danish patients referred to inor outpatient psychiatric hospital care and diagnosed with depression for the first time. It is often supposed, that the estimated prevalence of personality disorders among patients with depression can be influenced by the presence of depressive symptoms (Peselow et al., 1994), even though this has not been found by all authors (Loranger et al., 1991). The same concern may apply to the evaluation of life events due to recall bias. We conducted the interviews some time following discharge (median 147 days, quartiles 119–184 days), that is to say, apart from the acute depressive state, thereby probably reducing this source of error. Finally, we used comprehensive interviews to ensure a high validity of our data and a multidimensional approach, which enabled us to adjust for the possible confounding effects of personality factors and other variables. Since the genetic analyses were performed after the interviews, the investigators and the participants were blinded as to the genotypes. 4.3. Limitations Firstly, the retrospective evaluation of SLE is a potential source of error shared with the majority of previous studies that have undertaken the assessment of SLE during or after the depressive episode. However, there was no difference in severity of depressive symptoms between the groups with and without SLE and no effect of HDRS in the regression models. Further, since the genetic background was unknown, a misclassification of SLE according to genotype is likely to be random. Nevertheless, it cannot be excluded that the association between genotypes and SLE could be confounded by the severity of persisting depressive symptoms at the time of the interview. Secondly, we considered only recent SLE, since it has been shown, that the depressiogenic effect of environmental adversities is quickly declining with time (Kendler et al., 1998). We did not evaluate more distant SLE, chronic stress, childhood adversities, social support or positive life events, which might also interact with polymorphisms on depression or perhaps 3-way with genotype and recent SLE. Thirdly, we have evaluated SLE in a wide sense. It is possible that some subtypes of SLE exert a different influence on depression and show a different interaction with the genetic background. Fourthly, the method used in this study is suitable for evaluation of SLE × genotype interactions, but does not allow the estimation of the independent effects of exposure to SLE alone and the genotype alone. Fifthly, interpreting the results as a manifestation of genetic control of sensitivity to SLE (Fig. 1, pathway a) is based on the assumption, that exposure to SLE is independent from the genotypes of interest. Alternatively, is has been proposed that genes may act on the liability to depression by predisposing the individuals to select themselves into high risk environments, that is to say genetic control of exposure to SLE (Fig. 1, pathway e) (Kendler and Baker, 2007). Our data does not make discrimination between these two models possible (gene–environment interaction and

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gene–environment correlation, respectively). On the other hand, even though experience of environmental stressors may to some extend be heritable, it is less likely that the specific genotypes analysed in this study should influence the risk of experiencing SLE. Previous studies of 5-HTTLPR, which have reported no influence of genotype on exposure to stressors, support this assumption (Caspi et al., 2003; Kendler et al., 2005). Sixthly, the association between 5-HTTLPR and SLE did not withstand correction for multiple testing. Seventhly, we investigated a sample of patients referred to psychiatric hospital care, and therefore our findings cannot be generalized to patients with milder depressions treated in primary care. 4.4. Comparison with previous findings The majority of studies have principally replicated the interaction between SLE and 5-HTTLPR (Uher and McGuffin, 2008). However, some studies have found no gene–environment interaction on depression (Gillespie et al., 2005; Surtees et al., 2006; Chipman et al., 2007; Scheid et al., 2007; Covault et al., 2007; Power et al., 2008) or even an effect in the opposite direction (Sjoberg et al., 2006; Chorbov et al., 2007; Brummett et al., 2008) compared to the original findings by Caspi et al. (2003). The Met-allele of BDNF Val66Met, which has been associated with reduced BDNF activity (Egan et al., 2003), conferred increased vulnerability to depression in two investigations of depression following childhood adversities (Kaufman et al., 2006; Wichers et al., 2008) and one study of stressful life events among Korean elderly (Kim et al., 2007). One study of COMT (Mandelli et al., 2007) found a significant interaction with SLE on depression. The same study revealed no interaction between SLE and 5-HTR1A (Mandelli et al., 2007). The interactions of SLE with TPH1 and 5-HTR2C, respectively, have been evaluated in one study (Eley et al., 2004) with negative results. The effects of polymorphisms in ACE and 5-HTR2A have not been investigated. The majority of studies have either assessed depressive symptoms without establishing a diagnosis of depression or evaluated current or recent depressive episodes without taking into consideration the number of previous episodes. Caspi et al. (2003) assessed past-year depression at age 26 and SLE occurring between the 21st and the 26th birthday. In a subsequent analysis, the authors excluded individuals diagnosed with depression before age 21 (27%), nevertheless some of the past-year episodes at age 26 might still have been recurrences rather than first episodes of depression. No other study has included patients suffering exclusively from first episode depression, but three investigations (Gillespie et al., 2005; Wilhelm et al., 2006; Mandelli et al., 2007) have paid attention to the history of depression by asking the participants about SLE prior to the first depressive episode, even though some of them at the time of assessment had experienced one or more subsequent episodes. This method results in long time periods between onset of depression and the date of investigation and consequently an increased risk of recall bias. Further, the more imprecise dating of the first depressive episode as well as the occurrence of SLE implies a risk of confusing cause and effect. Among the investigations of SLE, four studies have used specific events (hip fracture (Lenze et al., 2005), AMI (Nakatani et al., 2005), hurricane exposure (Kilpatrick et al., 2007) and

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caregiver status (Brummett et al., 2008)). The remaining studies investigated broader categories of stressful life events assessed retrospectively during a time span of typically 6–12 months by means of a face-to-face interviews or questionnaires. Besides the study by Caspi et al. (2003), only five studies have evaluated the presence of SLE as well as the diagnosis of depression by way of a structured face-to-face interview. These five studies have all confirmed the interaction between 5-HTTLPR and SLE in predicting onset of depression (Kendler et al., 2005; Lenze et al., 2005; Wilhelm et al., 2006; Mandelli et al., 2007) or severity of depressive symptoms (Zalsman et al., 2006). Notable, the studies reporting no gene–environment interaction have all assessed both life events and depressive symptoms by means of questionnaires or telephone/lay interviews. Thus, the methodological differences seem to influence the results, suggesting that relatively smaller samples of more intensively evaluated participants might have some advantages when investigating gene–environment interactions compared to very large samples with data of less validity. Besides age and gender, the confounding or mediating effects of other variables have rarely been investigated. A few studies have adjusted for the effects of confounders such as family history of depression (Cervilla et al., 2007), marital status (Lazary et al., 2008), cognitive function (Kim et al., 2007), and social support (Kilpatrick et al., 2007). Further, the gene–environment interaction on depression could be mediated by personality factors such as the personality trait neuroticism, which has been associated both with depression (Kendler et al., 2006), the risk of exposure to SLE (Van et al., 2001; Kendler et al., 2003) and 5-HTTLPR (Sen et al., 2004). Nevertheless, only one study has considered the level of neuroticism (Jacobs et al., 2006), finding that the effect of SLE on depression was no longer dependent on 5-HTTLPR after taking neuroticism into account. We included neuroticism as well as comorbidity of personality disorder in the regression models and found on the contrary no effect of these variables, indicating a gene–environment interaction independent of personality factors. The influence of personality disorders on the gene–environment interaction has not been investigated in previous studies. 5. Conclusion The results indicate an interaction between SLE and the genotypes of 5-HTTLPR and BDNF Val66Met on first episode depression. In addition, we found no evidence that the gene– environment interaction was mediated by personality factors (neuroticism and personality disorder), no evidence for a 3way interaction between SLE, 5-HTTLPR and BDNF Val66Met, and no evidence for interactions between SLE and polymorphisms in COMT, TPH1, ACE, 5-HTR2A, and 5-HTR2C, respectively, on depression. The results add evidence to the opinion that genes influence the liability to depression not only by main effects on risk but also by control of sensitivity to the pathogenic effects of the environment. That view of the interplay between genetic risk and environmental stress might in the future direct preventive care. Nevertheless, a more comprehensive understanding of the role of genetic predictors of depression and their interactions with environmental factors is needed, before genetic testing can play a part in diagnosis, prediction, or prevention of depression.

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Role of funding source Nothing declared. Conflict of interest No conflict declared.

References Beck, A.T., Ward, C.H., Mendelson, M., Mock, J., Erbaugh, J., 1961. An inventory for measuring depression. Arch. Gen. Psychiatry 4, 561–571. Bondy, B., Kuznik, J., Baghai, T., Schule, C., Zwanzger, P., Minov, C., de, J.S., Rupprecht, R., Meyer, H., Engel, R.R., Eisenmenger, W., Ackenheil, M., 2000. Lack of association of serotonin-2A receptor gene polymorphism (T102C) with suicidal ideation and suicide. Am. J. Med. Genet. 96, 831–835. Brummett, B.H., Boyle, S.H., Siegler, I.C., Kuhn, C.M., shley-Koch, A., Jonassaint, C.R., Zuchner, S., Collins, A., Williams, R.B., 2008. 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, 34–43. 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, 386–389. Cervilla, J.A., Molina, E., Rivera, M., Torres-Gonzalez, F., Bellon, J.A., Moreno, B., Luna, J.D., Lorente, J.A., Mayoral, F., King, M., Nazareth, I., Gutierrez, B., 2007. The risk for depression conferred by stressful life events is modified by variation at the serotonin transporter 5HTTLPR genotype: evidence from the Spanish PREDICT-Gene cohort. Mol. Psychiatry 12, 748–755. Chipman, P., Jorm, A.F., Prior, M., Sanson, A., Smart, D., Tan, X., Easteal, S., 2007. No interaction between the serotonin transporter polymorphism (5-HTTLPR) and childhood adversity or recent stressful life events on symptoms of depression: results from two community surveys. Am. J. Med. Genet. B Neuropsychiatr. Genet. 144B, 561–565. Chorbov, V.M., Lobos, E.A., Todorov, A.A., Heath, A.C., Botteron, K.N., Todd, R.D., 2007. Relationship of 5-HTTLPR genotypes and depression risk in the presence of trauma in a female twin sample. Am. J. Med. Genet. B Neuropsychiatr. Genet. 144, 830–833. Christiansen, L., Tan, Q., Iachina, M., Bathum, L., Kruse, T.A., McGue, M., Christensen, K., 2007. Candidate gene polymorphisms in the serotonergic pathway: influence on depression symptomatology in an elderly population. Biol. Psychiatry 61, 223–230. Covault, J., Tennen, H., Armeli, S., Conner, T.S., Herman, A.I., Cillessen, A.H., Kranzler, H.R., 2007. Interactive effects of the serotonin transporter 5-HTTLPR polymorphism and stressful life events on college student drinking and drug use. Biol. Psychiatry 61, 609–616. Egan, M.F., Kojima, M., Callicott, J.H., Goldberg, T.E., Kolachana, B.S., Bertolino, A., Zaitsev, E., Gold, B., Goldman, D., Dean, M., Lu, B., Weinberger, D.R., 2003. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 112, 257–269. 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, 908–915. First, M.B., Gibbon, M., Spitzer, R.L., Williams, J.B.W., Benjamin, L.S., 1997. The Structured Clinical Interview for DSM — IV Axis II Personality Disorders (SCID — II). American Psychiatric Press, Washington. Gillespie, N.A., Whitfield, J.B., Williams, B., Heath, A.C., Martin, N.G., 2005. The relationship between stressful life events, the serotonin transporter (5-HTTLPR) genotype and major depression. Psychol. Med. 35, 101–111. Hamilton, M., 1960. A rating scale for depression. J. Neurol. Neurosurg. Psychiatry 23, 56–62. Jacobs, N., Kenis, G., Peeters, F., Derom, C., Vlietinck, R., Van, O.J., 2006. Stressrelated negative affectivity and genetically altered serotonin transporter function: evidence of synergism in shaping risk of depression. Arch. Gen. Psychiatry 63, 989–996. Kaufman, J., Yang, B.Z., Douglas-Palumberi, H., Grasso, D., Lipschitz, D., Houshyar, S., Krystal, J.H., Gelernter, J., 2006. Brain-derived neurotrophic factor-5HTTLPR gene interactions and environmental modifiers of depression in children. Biol. Psychiatry 59, 673–680. Kendler, K.S., Baker, J.H., 2007. Genetic influences on measures of the environment: a systematic review. Psychol. Med. 37, 615–626. Kendler, K.S., Karkowski, L.M., Prescott, C.A., 1998. Stressful life events and major depression: risk period, long-term contextual threat, and diagnostic specificity. J. Nerv. Ment. Dis. 186, 661–669. Kendler, K.S., Thornton, L.M., Gardner, C.O., 2000. Stressful life events and previous episodes in the etiology of major depression in women: an evaluation of the “kindling” hypothesis. Am. J. Psychiatry 157, 1243–1251.

Kendler, K.S., Thornton, L.M., Gardner, C.O., 2001. Genetic risk, number of previous depressive episodes, and stressful life events in predicting onset of major depression. Am. J. Psychiatry 158, 582–586. Kendler, K.S., Gardner, C.O., Prescott, C.A., 2003. Personality and the experience of environmental adversity. Psychol. Med. 33, 1193–1202. Kendler, K.S., Kuhn, J.W., Vittum, J., Prescott, C.A., Riley, B., 2005. The interaction of stressful life events and a serotonin transporter polymorphism in the prediction of episodes of major depression: a replication. Arch. Gen. Psychiatry 62, 529–535. Kendler, K.S., Gatz, M., Gardner, C.O., Pedersen, N.L., 2006. Personality and major depression: a Swedish longitudinal, population-based twin study. Arch. Gen. Psychiatry 63, 1113–1120. Khoury, M.J., Flanders, W.D., 1996. Nontraditional epidemiologic approaches in the analysis of gene–environment interaction: case–control studies with no controls! Am. J. Epidemiol. 144, 207–213. Kilpatrick, D.G., Koenen, K.C., Ruggiero, K.J., Acierno, R., Galea, S., Resnick, H.S., Roitzsch, J., Boyle, J., Gelernter, J., 2007. The serotonin transporter genotype and social support and moderation of posttraumatic stress disorder and depression in hurricane-exposed adults. Am. J. Psychiatry 164, 1693–1699. Kim, J.M., Stewart, R., Kim, S.W., Yang, S.J., Shin, I.S., Kim, Y.H., Yoon, J.S., 2007. Interactions between life stressors and susceptibility genes (5-HTTLPR and BDNF) on depression in Korean elders. Biol. Psychiatry 62, 423–428. Lazary, J., Lazary, A., Gonda, X., Benko, A., Molnar, E., Juhasz, G., Bagdy, G., 2008. New Evidence for the association of the serotonin transporter gene (SLC6A4) haplotypes, threatening life events, and depressive phenotype. Biol. Psychiatry. Lenze, E.J., Munin, M.C., Ferrell, R.E., Pollock, B.G., Skidmore, E., Lotrich, F., Rogers, J.C., Quear, T., Houck, P., Reynolds III, C.F., 2005. Association of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) genotype with depression in elderly persons after hip fracture. Am. J. Geriatr. Psychiatry 13, 428–432. Loranger, A.W., Lenzenweger, M.F., Gartner, A.F., Susman, V.L., Herzig, J., Zammit, G.K., Gartner, J.D., Abrams, R.C., Young, R.C., 1991. Trait-state artifacts and the diagnosis of personality disorders. Arch. Gen. Psychiatry 48, 720–728. Mandelli, L., Serretti, A., Marino, E., Pirovano, A., Calati, R., Colombo, C., 2007. Interaction between serotonin transporter gene, catechol-O-methyltransferase gene and stressful life events in mood disorders. Int. J. Neuropsychopharmacol. 10, 437–447. Munk-Jorgensen, P., Mortensen, P.B., 1997. The Danish psychiatric central register. Dan. Med. Bull. 44, 82–84. 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, 32–38. Nakatani, D., Sato, H., Sakata, Y., Shiotani, I., Kinjo, K., Mizuno, H., Shimizu, M., Ito, H., Koretsune, Y., Hirayama, A., Hori, M., 2005. Influence of serotonin transporter gene polymorphism on depressive symptoms and new cardiac events after acute myocardial infarction. Am. Heart J. 150, 652–658. Paykel, E.S., 1997. The interview for recent life events. Psychol. Med. 27, 301–310. Peselow, E.D., Sanfilipo, M.P., Fieve, R.R., Gulbenkian, G., 1994. Personality traits during depression and after clinical recovery. Br. J. Psychiatry 164, 349–354. Post, R.M., 1992. Transduction of psychosocial stress into the neurobiology of recurrent affective disorder. Am. J. Psychiatry 149, 999–1010. Power, T., Stewart, R., Ancelin, M.L., Jaussent, I., Malafosse, A., Ritchie, K., 2008. 5-HTTLPR genotype, stressful life events and late-life depression: no evidence of interaction in a French population. Neurobiol. Aging. Praschak-Rieder, N., Kennedy, J., Wilson, A.A., Hussey, D., Boovariwala, A., Willeit, M., Ginovart, N., Tharmalingam, S., Masellis, M., Houle, S., Meyer, J.H., 2007. Novel 5-HTTLPR allele associates with higher serotonin transporter binding in putamen: a [(11)C] DASB positron emission tomography study. Biol. Psychiatry 62, 327–331. Rasmussen, H.B., Werge, T.M., 2007. Novel procedure for genotyping of the human serotonin transporter gene-linked polymorphic region (5HTTLPR) — a region with a high level of allele diversity. Psychiatr. Genet. 17, 287–291. Scheid, J.M., Holzman, C.B., Jones, N., Friderici, K.H., Nummy, K.A., Symonds, L.L., Sikorskii, A., Regier, M.K., Fisher, R., 2007. Depressive symptoms in mid-pregnancy, lifetime stressors and the 5-HTTLPR genotype. Genes Brain Behav. 6, 453–464. 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, 85–89. 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, 443–449.

J. Drachmann Bukh et al. / Journal of Affective Disorders 119 (2009) 107–115 Strobel, A., Gutknecht, L., Rothe, C., Reif, A., Mossner, R., Zeng, Y., Brocke, B., Lesch, K.P., 2003. Allelic variation in 5-HT1A receptor expression is associated with anxiety- and depression-related personality traits. J. Neural Transm. 110, 1445–1453. Surtees, P.G., Wainwright, N.W., Willis-Owen, S.A., Luben, R., Day, N.E., Flint, J., 2006. Social adversity, the serotonin transporter (5-HTTLPR) polymorphism and major depressive disorder. Biol. Psychiatry 59, 224–229. Uher, R., McGuffin, P., 2008. The moderation by the serotonin transporter gene of environmental adversity in the aetiology of mental illness: review and methodological analysis. Mol. Psychiatry 13, 131–146. Van, O.J., Park, S.B., Jones, P.B., 2001. Neuroticism, life events and mental health: evidence for person–environment correlation. Br. J. Psychiatry (Suppl 40), s72–s77. Wichers, M., Kenis, G., Jacobs, N., Mengelers, R., Derom, C., Vlietinck, R., Van, O.J., 2008. The BDNF Val(66)Met x 5-HTTLPR x child adversity interaction

115

and depressive symptoms: an attempt at replication. Am. J. Med. Genet. B Neuropsychiatr. Genet. 147B, 120–123. Wilhelm, K., Mitchell, P.B., Niven, H., Finch, A., Wedgwood, L., Scimone, A., Blair, I.P., Parker, G., Schofield, P.R., 2006. Life events, first depression onset and the serotonin transporter gene. Br. J. Psychiatry 188, 210–215. Wing, J.K., Babor, T., Brugha, T., Burke, J., Cooper, J.E., Giel, R., Jablenski, A., Regier, D., Sartorius, N., 1990. SCAN. Schedules for clinical assessment in neuropsychiatry. Arch. Gen. Psychiatry 47, 589–593. World Health Organisation, 1992. The ICD-10 Classification of Mental and Behavioural Disorders: Clinical Descriptions and Diagnostic Guidelines. World Health Organisation, Geneva. Zalsman, G., Huang, Y.Y., Oquendo, M.A., Burke, A.K., Hu, X.Z., Brent, D.A., Ellis, S.P., Goldman, D., Mann, J.J., 2006. Association of a triallelic serotonin transporter gene promoter region (5-HTTLPR) polymorphism with stressful life events and severity of depression. Am. J. Psychiatry 163, 1588–1593.