The moderation by the serotonin transporter gene of environmental adversity in the aetiology of mental illness: review and methodological analysis

Abstract

Gene–environmental interaction (G × E) between a common functional polymorphism in the promoter region of the serotonin transporter gene (5-HTT) and environmental adversity on the onset of depression in humans has been found in fifteen independent studies. It is supported by evidence from animal experiments, pharmacological challenge and neuroimaging investigations. However, negative findings have been reported in two large samples. We explore reasons for the inconsistencies and suggest means to their resolution. Sample age and gender composition emerge as important factors. While the G × E has been consistently detected in young adult samples, there are contradictory findings in adolescent boys and elderly people. The method of assessment of environmental adversity is also important with detailed interview-based approaches being associated with positive G × E findings. Unresolved issues in the definition of the genotype include the dominance of alleles and influence of other polymorphisms, both in 5-HTT and other genes. Assessment of multiple adverse outcomes, including depression, substance use and self-destructive behaviour is needed to clarify the generalisability of the G × E pathogenic mechanisms. Biological and behavioural intermediate phenotypes are yet to be exploited to understand the mechanisms underlying the G × E.

Introduction

Research on genetic factors in mental illness has been marked by a discrepancy between high heritability estimates and a paucity of replicable gene-disorder associations.1 One explanation for this incongruity is that at least some specific gene effects are conditional on environmental factors, that is, gene–environment interaction (G × E) is present.2, 3

Quantitative human population genetics studies indicate that G × E plays a role in the development of depression.4, 5, 6, 7 In the last five years, G × E involving specific gene polymorphisms has been identified in animals8, 9 and humans.10, 11, 12, 13 Encouragingly, replications14, 15 and a positive metaanalysis16 indicate that measuring environment improves the reliability of genetic research.

In 2003, Caspi et al.11 reported that carriers of the short (‘s’), transcriptionally less active, allele of common functional polymorphism (5–HTTLPR) in the promoter of the serotonin transporter gene (5-HTT) are more likely to develop depression following stressful life events or childhood abuse than individuals homozygous for the long (‘l’) allele. This demonstration of G × E has attracted a large number of replication attempts that have used varied methodologies in diverse samples. We review these studies and explore methodological aspects possibly explaining the heterogeneity of findings. We conclude with recommendations for future research.

Animal models

In mouse, there is no analogue to the human 5-HTTLPR polymorphism, but it is possible to either disable the 5-HTT gene or to transgenically increase its expression.17, 18 5-HTT knockout mice show behaviours consistent with anxious and depressive traits; they appear reluctant to explore brightly lit spaces or elevated open platforms and give up struggling early when put in an uncomfortable situation.19, 20, 21 Interestingly, the difference from wild-type animals in depression-like giving-up (‘behavioural despair’) is manifest only after repeated exposure to stressors, analogous to repeated or chronic stressful life events in humans.20 The 5-HTT knockout animals also show exaggerated neuroendocrine reactions to acute stress similar to the HPA axis over reactivity in depressed humans.22, 23 The effect of 5-HTT knockout on anxiety- and depression-like behaviours may be mediated by the absence of functional serotonin clearance mechanism during a vulnerable developmental period,24 but can still be selectively reversed by pharmacological 5HT1A receptor blockage later in life suggesting a life-long modulatory involvement of the serotonergic system.19 The anxious-depressive phenotype in 5-HTT knockout mice is associated with increased dendritic branching in the ‘fear circuit’ including the amygdala and limbic cortex.20 On the other hand, mice with transgenically increased 5-HTT expression appear fearless and explore bright-lit and open spaces more daringly than wild-type animals.17

Given the importance of early life stressors and their interaction with 5-HTTLPR genotype in the development of psychiatric disorders in humans, it would be of interest to model the interaction of 5-HTT knockout with early life stress in mice. However, the 5-HTT knockout status does not interact with exposure to mild postnatal foot-shock stress.25 Interactions with more chronic and severe forms of stress, such as maternal separation, remain to be explored. Thus, while the mouse knockout model confirms the association of 5-HTT with anxious and depressive traits and impaired adaptation to repetitive stress it has so far not provided evidence supporting the interaction with early life stressors.

In the rhesus macaque, there is a length polymorphism in the 5-HTT promoter region functionally analogous to the human 5-HTTLPR.9 The less active s-allele interacts with adverse early rearing conditions (peer rearing) to result in more distress, less activity, stronger neuroendocrine responses to stress, higher consumption of alcohol and lower serotonin turnover in the central nervous system.8, 9, 26, 27 During peer-rearing, animals homozygous for the l allele develop more socially acceptable playful behaviours but sl heterozygotes tend to become aggressive, suggesting that the ll homozygous status confers an adaptive potential for adverse environment.28 In conclusion, the non-human primate experiments support the G × E hypothesis by showing that the s allele is associated with a vulnerability to early life adverse circumstances leading to multiple adverse outcomes resembling human psychiatric disorders.

Human studies

To date, seventen studies have been published that have attempted to extend and replicate the findings of Caspi et al. (Table 1). Taken at face value, there were eleven replications,15, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 three partial replications in females only,40, 41, 42 and three non-replications.43, 44, 45 Importantly, two studies with very large samples were negative.44, 45 On the other hand, some ‘positive’ studies have been published with samples so small that they cannot be considered to test the hypothesis.46, 47 This could suggest a publication bias with smaller negative studies remaining unpublished or could be explained by other systematic differences between negative and positive studies. The replication studies have used varied designs, samples and measures, precluding direct comparison and meta-analysis. However, the diversity of methodology and findings provides an opportunity to explore the G × E in detail, specify methodological requirements and form specific hypotheses.

Table 1 Summary of human studies of the interaction of the 5-HTTLPR with environmental adversity on the onset or severity of depression

Study design

Prospective cohort study design has been used in seven studies;11, 15, 29, 42, 43, 44 although these studies were prospective in nature and included repeated assessments, the depression and SLE were typically assessed at the same time point, leaving the possibility that recall of SLEs may be influenced by the depressed state.48 Six studies were cross-sectional surveys.30, 31, 34, 37, 39, 41, 45 One study used a combined cohort/case–control design, selecting participants with extreme scores on either environmental adversity or depression for genotyping.40 One study examined individuals exposed to a major stressor (myocardial infarction).38 Two studies used cases-only design to investigate the association of SLE preceding the onset of depression with genotype49 or the interaction of SLE during the course of the disorder with genotype on current symptom severity.36 There is no obvious association between study design and results.

The study by Caspi and colleagues11 employed several methodological features that make causal inferences plausible: (a) informant report of depression symptoms and multiple sources to identify environmental stressors were used to obviate common source information bias; (b) the temporal order of events was established by demonstrating that past 6 year SLEs are more strongly associated with past 12 months depression than with depression preceding the SLE reporting period. These features were not applied in the replication studies.

The two large negative studies merit attention. In the cross-sectional study by Surtees et al. (2006), SLEs and depression symptoms were self-reported as part of the same questionnaire, the psychometric properties of which are not reported. In the study by Gillespie and colleagues,44 SLEs and depressive symptoms were reported in a questionnaire and the diagnosis of depression was assessed retrospectively by interview conducted 4 years later. The weak or non-significant associations between SLEs and depression in these studies suggest problems with the validity of self-report measures of SLE50 and, in the study by Gillepie and colleagues44 possible inaccuracies of the retrospective assessment of depression.51

Sample, age and gender

Four studies used community-based population samples or unselected birth cohorts,11, 32, 41, 42 three studies were conducted on twin registers,15, 29, 44 one used unselected primary care attendees,37 three studies used convenience samples of students or teachers;34, 35, 43 three studies used enriched samples selected based on experience of childhood abuse,31 high levels of social adversity and depression40 or extremes on neuroticism45 and two studies used clinical samples of depressed patients.33, 36

Sample size is a major determinant of power to detect G × E.52 It is therefore notable that the two studies with largest samples produced negative findings.44, 45 However, both of these samples were recruited and assessed for reasons other than depression research: their primary aims were study of cancer45 or addiction44 and the assessment procedures were not designed for the study of depression and SLEs. It has been demonstrated that inaccurate measurements decrease power so substantially that smaller samples with accurate assessments are preferable.53

The incidence and aetiology of depression varies by age and gender,54 hence it is germane to explore whether the G × E interaction is specific to certain age or/and gender group. Across the studies exploring the 5-HTTLPR mediation of environmental adversity in the aetiology of depression, age appears to be a major factor, potentially accounting for substantial proportion of the heterogeneity in results. The one study in children shows interaction effect consistent with the young adult studies.31 The two adolescent studies both show an interaction in the expected direction among females only and trend for an opposite effect in males.40, 42 The G × E interaction is consistently positive in young adults.11, 15, 29, 33, 35 Note that in the study of Mandelli and colleagues33 the middle-aged participants are retrospectively interviewed about events preceding their first affective episode, which occurred on average in their early 30s. Even in a study where positive results were reported separately for both genders, the G × E was stronger in females.15 Of the four studies with predominantly middle-aged to elderly samples, two showed no evidence of interaction with the non-significant interaction signs pointing in the opposite direction;44, 45 one identified an interaction in the expected direction for female participants only,41 and one in the whole sample after correcting for gender.32

The observed pattern of variation by age and gender is likely to reflect several interacting factors. Depression is more common in females.55 The depressogenic effect of life events as measured by interview or self-report is stronger in females than in males, especially during adolescence.56, 57 It is possible that the current instruments do not give sufficient weight to life events relevant for adolescent males (for example events related to peer prestige) or that alternative outcomes, such as substance abuse or antisocial behaviour substitute depression in males.58, 59, 60 The apparent effect of age may also reflect the distinction between first episode and recurrence of depression as the effect of SLEs decreases with the number of episodes.61 For example, in the sample of Surtees et al.45 90% of depressive episodes were recurrences rather than first episodes, partially explaining the small size of SLE main effect and negative finding regarding G × E interaction.

Life events are common and most people encounter at least one severe SLE by reaching middle age. Therefore, if a stable individual vulnerability to the depressogenic effect of life events is present, it is likely to manifest at an earlier rather than later age. Indeed, as exposure to SLEs is nearly universal, it would be expected that with advancing age, the G × E gradually transforms into a direct gene-disorder association, especially for lifetime diagnosis of depression. However, no such direct association was reported in the older samples.32, 41, 44, 45 Therefore, the alternative explanation should be considered that the 5-HTTLPR genotype-mediated vulnerability operates in a developmental period comprising young adulthood.

Finally, we would like to consider the effect of selecting samples based on the extremes of environmental adversity or neuroticism. Because there is a strong positive correlation between depression and neuroticism,62 large proportion of variance in depression in such extreme sample will be explained by neuroticism alone. The G × E interaction may have strongest effect on depression among those with average levels of neuroticism, who are not represented in the extreme sample. Similar effect has been observed for SLE, with mild SLEs showing greater 5-HTTLPR mediation than severe SLEs in their depressogenic effect.15 Therefore, we propose that extreme distribution of neuroticism together with older age of subjects may explain the negative result of the study by Surtees et al.45

Definition of genotype

5-HTTLPR long and short alleles

The serotonin transporter (5-HTT) is located at the presynaptic membranes of serotonergic nerve terminals where it performs sodium-dependent transport of the neurotransmitter serotonin from the synaptic cleft back to the nerve terminal, thus limiting the action of serotonin to a short period after its release. Several lines of research converge to make the 5-HTT gene (termed SLC6A4; solute carrier family 6, member 4) a prime candidate gene for studies on the aetiology of depression: (a) the role of serotonin in the regulation of mood, activity, sleep and appetite,63 (b) the development of depression-like features in animals after disruption of the 5-HTT function in the neonatal period,24 (c) the efficacy of 5-HTT pharmacological inhibition to alleviate depression,64 (d) the provocation of depressive symptom relapse by depletion of the serotonin precursor tryptophan.65

Several polymorphisms in the SLC6A4 gene have been described, including a variable number tandem repeat in exon 2,66 a restriction fragment-length polymorphism in the 3′ untranslated region,67 and a variable number tandem repeat polymorphism in the 5′ promotor region, termed the 5-HTT gene-linked polymorphic region (5-HTTLPR).68 The 5-HTTLPR has two common alleles: a ‘long’ (l) allele with 16 imperfect repeats of an approximately 22-base-pair element and a ‘short’ (s) allele with 14 repeats.

The functionality of 5-HTTLPR

The 5-HTTLPR has attracted most research interest because it is common in human populations of all ethnicities,69 and it appears to have impact on 5-HTT gene expression and serotonergic system function.

In vitro studies of human lymphoblastoid cells show that the 5-HTTLPR s allele has lower transcriptional efficiency than the l allele at baseline and after stimulation.68, 70, 71, 72, 73 However, the translation of in vitro findings into in vivo functioning does not appear straightforward. Human neuroimaging studies of 5-HTT availability74, 75, 76, 77, 78, 79 and post-mortem studies of 5-HTT bindings and rRNA expression80, 81, 82 have not detected any consistent effect of 5-HTTLPR. No reliable association was found between 5-HTLPR and the level of serotonin metabolite in the cerebrospinal fluid.83, 84 Lower availability of the 5-HT1A receptor in 5-HTTLPR s allele carriers has been reported and awaits replication.85

While single measurements of 5-HTT availability and serotonin turnover have not provided a consistent picture, specific challenge tests may be better suited to reveal differences in central nervous reactivity associated with 5-HTTLPR. For example, most neuroendocrine tests of central serotonergic function have shown blunted responses in s allele carriers; these include smaller prolactin increases in response to fenfluramine, clomipramine or citalopram.86, 87, 88 On the other hand, the prolactin response to meta-chlorphenylpiperazine (m-CPP) was enhanced in ss homozygotes.49 Unlike fenfluramine, clomipramine and citalopram, which act directly on the serotonin transporter to increase extracellular serotonin, the m-CPP is a postsynaptic 5-HT agonist, inducing prolactin release via its action on the 5-HT2C receptors. Thus, the observed pattern of findings in ss carriers could reflect decreased presynaptic 5-HTT (explaining blunted reaction to fenfluramine, clomipramine or citalopram), but increased postsynaptic 5-HT2C receptor sensitivity (responsible for increased reaction to m-CPP). However, the results with m-CPP are based on a single study and 5-HT2C receptor function has not been directly investigated in relation to 5-HTTLPR.

Tryptophan depletion

A promising experimental measure of serotonergic function with direct relevance to depression is mood change during tryptophan depletion. Tryptophan, the precursor of serotonin, competes for transport into the central nervous system with other amino acids. In tryptophan depletion, human volunteers ingest large amount of amino acids other than tryptophan. This decreases the availability of tryptophan and subsequently of serotonin within several hours. During the depletion period, a proportion of individuals at risk for major depression experience reversible mood deterioration and other symptoms of depression, including psychomotor slowing, pessimism and impaired insight.65 Several studies indicate that the 5-HTTLPR contributes to the individual variability in mood response to tryptophan depletion. Among individuals with a history of depression, the ll phenotype is consistently associated with more severe mood deterioration during tryptophan depletion.89, 90 On the other hand, among individuals without personal history of depression, the effect of TD is smaller, includes changes in anxiety, punishment sensitivity and motivation rather than mood and is more pronounced among ss homozygotes.91, 92, 93, 94 Neumeister and colleagues90 proposed a model explaining this apparent interaction of 5-HTTLPR with personal history of depression by differential activity of pre- and postsynaptic 5-HT1A and 5-HT1B receptors.

Genetic neuroimaging

Neuroimaging techniques including the functional magnetic resonance imaging (fMRI) allow flexible and non-invasive exploration of human brain function in vivo. However, the fMRI method is limited by low signal-to-noise ratio and the absence of true baseline, which complicate the interpretation of results. Human fMRI studies indicate replicable association between the 5-HTTLPR s allele and greater reactivity to negative emotional compared to neutral stimuli in the amygdale.95, 96, 97, 98 This association is independent of gender and the temperamental trait of harm avoidance.96 Furthermore, the ‘functional connectivity’ (correlation of activity) of the amygdala to the ventromedial prefrontal cortex was stronger98 and the connectivity to the perigenual anterior cingulate was weaker99 in the s allele carriers compared to ll homozygotes.98, 99 The relative ‘uncoupling’ of amygdala from the anterior cingulate in the s allele carriers correlated with a measure of harm avoidance, suggesting that strong amygdala activity free from anterior cingulate control may be responsible for anxiety-related traits associated with the 5-HTTLPR.99

Studies that include a passive baseline (fixation cross) show that the apparent over-reactivity to negative stimuli is largely explained by decreased reactivity of the amygdala to neutral stimuli compared to the passive baseline.100, 101 This pattern of findings may reflect an increased resting amygdala activity in s allele carriers, but this would have to be tested using a method with an absolute baseline. Using MRI perfusion imaging, Canli and colleagues found support for increased resting activity in the left amygdala and the hippocampus.102 Similar baseline functional differences are suggested by an acoustic startle study showing that s allele carriers have stronger baseline startle reaction, but do not differ in emotional startle potentiation from ll homozygotes.103 Furthermore, a whole brain exploration indicates widespread structural and functional differences between s allele carriers and ll homozygotes in other brain regions: the s allele appears to be associated with reduced volume and grey matter density in several frontal-lobe regions, greater reactivity to negative emotional stimuli in the insula and striatum and stronger reactivity to positive emotional stimuli in the left frontal and posterior cingulate regions.100 These results, contingent on replication, indicate that the 5-HTTLPR may have a pervasive effect on brain structure and function.

As a more direct functional probe of the G × E interaction, Canli and colleagues102 explored the interacting effects of stressful life events and 5-HTTLPR genotype on the brain function at rest and in reaction to emotional stimuli. They found that number of stressful life events was associated with higher left amygdala and hippocampus perfusion at rest, and lower relative reactivity to neutral stimuli in s allele carriers compared to ll homozygotes. Thus it appears that the brain reactivity is differentially shaped by experience depending on genetic background rather than representing an ‘endophenotype’ independent of environment.

Allelic variants of 5-HTTLPR: LA and LG or rs25531

Other less common alleles of the 5-HTTLPR have been described in Asian populations.69, 104 An adenosine/guanine (A/G) single-nucleotide polymorphism (SNP) located within the repeats of 5-HTTLPR seems to be relatively common even in Caucasian populations, with a minor (G) allele frequency (MAF) of 10%; it has been described as rs25531 SNP associated with either short or long 5-HTTLPR alleles105 or as a LA/LG modifier of the long allele.106 Wendland and colleagues107 have demonstrated that the disagreement was caused by two alternative demarcations of the 5-HTTLPR insertion sequence and that the G/A rs25531 SNP can be associated with either l or s allele. It was reported, that in lymphoblastoid cell lines, the S and LG alleles were associated with lower 5-HTT expression compared to the LA allele.106 However, this finding has not been consistently replicated.108 It is also unclear whether the rs25531 SNP has any impact on the functionality of the short 5-HTTLPR allele.

On the assumption that the in vitro 5-HTT expression determines the functionality of alleles in humans, the distinction between LA and LG alleles has been applied to the G × E research, with S and LG being coded as ‘low expressing alleles’ and contrasted to the LA ‘high expressing allele.’36 However, it is presently unknown whether the in vitro transcriptional activity differences are responsible for the G × E interaction observed at the population level. We suggest that the two models are compared in future investigations; if the reported in vitro functionality determines the G × E, the specification of LA and LG alleles should improve model fit. Given that the rs25531 SNP can combine with either s or l allele, the two polymorphisms can be considered independently for analysis purposes.107

The role of heterozygosity

While most studies agree on the differential environmental effects on ss versus ll homozygotes, the findings in and reporting of effects in sl heterozygotes have been less consistent. Dominant, co-dominant and recessive models of the 5-HTTLPR s allele effect have all been proposed, but there is no clear consensus. We review here the evidence from animal and human, laboratory and observational studies relevant to this issue.

In 5-HTT knockout mice, the heterozygotes tend to show intermediate levels of anxiety-like behaviours consistent with a co-dominant (additive) effect; additionally heterozygotes are free of gross developmental anomalies that could affect such behavioural tests.109, 110 Non-human primate models are restricted to heterozygotes as no studies in ss homozygotes are available.

The first gene-expression study in human lymphoblastoid cells contraintuitively suggested a dominant effect of the less expressing s allele on 5-HTT production.72 However, an additive co-dominant effect was demonstrated in larger samples considering s/l70 or S, LA/LG alleles.106 In contrast, human functional neuroimaging studies suggest functionally dominant s allele.96, 98

The human G × E studies provide an inconsistent picture. In the original investigation by Caspi and colleagues,11 the data pattern is clearly indicative of a co-dominant effect with the s/l heterozygotes falling intermediate in all analyses and similar pattern was reported in several other studies.29, 35 A study in elderly Korean sample found G × E results consistent with dominant s allele.32 Some authors assumed a dominant s based on an early 5-HTT expression study and did not report results separately for heterozygotes.41 On the other hand, Kendler and colleagues15 found that a model with recessive s (that is with ll and ls combined) provided best fit to their data and at least one other study showed results consistent with recessive s.34 Results from child and adolescent samples place heterozygotes intermediate but close to ll homozygotes, suggesting a predominantly recessive s effect for early-onset depression.30, 40, 42 When multiple outcomes were considered within one sample, a co-dominant effect was shown for the more common outcome of alcohol abuse, but a recessive s effect was apparent for the less common illicit drug use.43

In conclusion, the role of 5-HTTLPR heterozygosity in G × E remains unclear. It is possible that the distinction is quantitative: while effect on softer outcomes may be co-dominant or recessive, more severe outcomes and early onset may be associated with an ss homozygote status. In future studies, it will be most important to report findings separately for each genotype and avoid assumptions based on selected literature. Complete reporting of descriptive statistics by genotype will enable meta-analyses addressing the issue.

Lesson from pharmacogenetics

Pharmacogenetic investigations involving the 5-HTTLPR have brought some intriguing results, relevant to the G × E research. An initial European investigation showed that among sufferers of major depression, 5-HTTLPR long allele is associated with better response to serotonergic antidepressants.111 This finding has been followed by several replications in European samples.112, 113 However, findings in Asian and American samples have been inconsistent, including findings of effect in the opposite direction in Korean samples114, 115 and of no association with antidepressant response in a large multicentre USA study.116 These differential findings suggest that there is no direct association of antidepressant repose and the 5-HTTLPR. Rather, the apparent association may be a marker of other factors, such as polymorphisms in population-specific linkage disequilibria with the 5-HTTLPR. Given this insight, the findings of G × E on population level should not be automatically considered to be due to the functional 5-HTTLPR unless a direct relationship is clearly demonstrated.

There is also uncertainty as to the nature of the short allele effect in pharmacogenetics studies of antidepressant response with different studies suggesting recessive,111, 112 dominant117 or co-dominant effects118 or even a heterosis effect with worst response in the heterozygotes.119

Epistasis with the brain-derived neurotrophic factor (BDNF)

It is unlikely that the 5-HTTLPR interacts with environmental factors independently of other genes. The possibility of epistatic effects and gene × gene × environment interactions opens a broad avenue for exploration with the hope to explain larger proportion of the observed variability in response to environmental factors. Given the shear number of potential interactions and statistical power limited by the size of available samples, it appears reasonable to first focus such explorations on biologically plausible candidate genes.

The BDNF emerged as a first candidate for epistasis with 5-HTTLPR. BDNF is one of the neurotrophic factors, with an important role in brain development and activity-dependent plasticity. It is produced and secreted as a longer pro-BDNF, which is then trimmed by proteases to the mature 153-amino-acid BDNF that binds to tyrosine-kinase receptor Trk B to influence gene expression in the target cell. Interaction between BDNF and 5-HT function has been demonstrated in animal models. BDNF heterozygous knockout mice show disturbances in serotonergic function including premature age-associated decrease in forebrain 5-HT.120 Interacting BDNF and 5-HTT effects on brain monoamine levels, stress response and anxiety-like behaviour were demonstrated in double-mutant male mice lacking one BDNF and both 5-HTT alleles.121 Interestingly, female gender or oestrogen administration protect against most neurochemical and behavioural effects of the combined 5-HTT/BDNF knock out.122

In humans, there is a common functional val66met (rs6265) single-nucleotide polymorphism in the translated region, which is part of the pro-BDNF, but not the mature BDNF. The less common met allele (allele frequency 19% in Caucasians and 44% in Asians) is associated with less efficient intracellular pro-BDNF trafficking, impaired activity-dependent BDNF secretion, smaller hippocampal grey matter volume and poor episodic memory.123 The met allele has been associated with increased risk to psychiatric disorders, including eating disorders and schizophrenia but not mood disorders.124

Two 5-HTTLPR G × E studies with samples at the extremes of the age span have reported a three-way interaction between environmental adversity, the 5-HTTLPR and BDNF val66met polymorphism.31, 32 In both cases, the three-way interaction including BDNF was stronger than the two-way interaction of SLE with 5-HTTLPR. The BDNF genotype was not included in the other 5-HTTLPR G × E studies, and to our knowledge, no negative findings have been reported so far. Given the strong rationale from animal studies and agreement of findings from two very different human populations, it appears worthwhile exploring the combined BDNF and 5-HTTLPR genotype in available samples.

Environmental adversity and stressors

There are a number of established environmental risk factors for depression. The studies of G × E in depression used stressful life events,11, 15, 29, 32, 33, 35, 43, 44 childhood abuse,11, 31, 45, 34 chronic somatic illness41 and composite indexes of environmental adversity.40, 42 Moffitt et al.3 specified that a candidate environmental pathogen for G × E should be a plausible cause of the disorder, proximal to the disorder onset, and have variable effect on different individuals; they further stipulated that cumulative, repeated, or protracted exposures are stronger pathogens than discrete acute events. The stressful life events fit these criteria; childhood abuse is probably not a proximal factor except in childhood depression; composite indexes of adversity are inclusive and cumulative, but it is difficult to date these and establish how close their temporal and aetiological association with the onset of depression is. The proximity criterion should be understood in the aetiological rather than strictly temporal sense and initiatives have been set up to encourage the search for aetiologically most relevant aspects of environmental adversity to streamline the G × E research.125

Research into the role of stressful life events and difficulties in the causation of depression has a long history.126, 127 It has been established that stressful life events (SLE) have a causal role in the genesis of depression,128 their depressogenic effect is variable and depends on the level of ‘contextual threat’,126 they are closely related in time to the onset of depression129, 130 and have stronger effect on first onset of depression than on recurrences.61 The effect of SLE is not specific to depression as they are also associated with the onset of anxiety disorders,131 substance abuse,132 eating disorders133 and psychosis.134

Assessment of SLE

The assessment of SLE is almost always retrospective and it is crucial to minimise recall bias, distortions and inaccuracies. The recall of SLEs is influenced by the depressed state and can be experimentally manipulated by mood induction.135 The SLEs and their contextual threat are most accurately assessed by structured interviews, which are preferable to self-report questionnaires.126, 136 However, the only way to obviate the retrospective recall bias is to obtain an objective record of the SLE.137

Of the reviewed studies, five used structured interviews,11, 15, 33, 35, 36 one used semistructured interview,42 nine used questionnaires29, 32, 34, 37, 40, 41, 43, 44, 45 and one used objective record of child abuse.31 All studies that used a structured interview to assess SLEs have found a G × E with the 5-HTTLPR s allele. All three negative studies used self-report questionnaires. The main effect of the SLE on depression was smaller in studies that failed to replicate the 5-HTTLPR G × E44, 45 than in studies that showed positive results. One of the negative 5-HTTLPR G × E studies44 reported a stronger main effect of SLE on depression onset before than after SLE reporting period, suggesting reversed causality. In conclusion, the varied methodology of SLE assessment in G × E studies may explain the inconsistent results. It is possible that the type of SLE that interacts with 5-HTTLPR genotype is not reliably reported in the self-report instruments and requires structured interview approach to be elicited.

Temporal relationship between SLE and depression onset

The temporal relationship between SLE and depression onset is incompletely understood. The effect of SLE on depression onset tends to decay over several months, with strongest effect in the month immediately following the SLE.129, 137 However, the temporal relationship varies by type of event; while job loss or physical illness have short-lived effects waning within several months, the depressogenic effects of legal problems or a loss of confidant are undiminished or even growing after 6 months.129 SLE reported at age 16 still predict depression six years later.138 The duration of the depressogenic effect of SLE also differs by gender: effect of marital loss decays with a half-life of 5 months in women and half-life of 21 months in men.130

In the 5-HTTLPR G × E studies, the time period for recording life events varied from 3 months15 to 5 years.11 In one small G × E study, the G × E interaction was significant for SLEs over five years but not for SLEs in the one year preceding the depression onset.35 Several researchers have pointed out that the long reporting period of SLEs in some of the G × E studies make a direct interaction between genes and the SLEs an unlikely explanation for the observed effect.29 This issue would best be explored by following a cohort of participants for a sufficient period of time after an objectively recorded independent stressful event and establishing the time course of G × E in relation to the main effect of the SLE. Given the observed gender differences in the time relationship between SLE and depression onset, gender should always be included in the analysis.

Genetic control of the environment

In the context of genetic research, it is noteworthy that experience and recall of SLE are partially under the control of genes.4, 139, 140, 141, 142 Neuroticism and personal history of depression predict reporting of SLE.143, 144 The genetic factors that influence retrospective report of environmental factors overlap with those that affect personality.141 However, the distinction of dependent and independent SLEs128, 145 and use of objectively recorded SLE137 have demonstrated that there is a causal relationship between SLE and depression, independent of individual differences in personality and recall. Unfortunately, the studies of 5-HTTLPR G × E and depression so far published have not used such methodologies and therefore the alternative explanation remains a possibility that a stable trait associated with experience and report of SLEs could explain what appears to be a G × E.29

Stressful life or neurotic trait?

Jacobs and colleagues29 have suggested that rather than SLE, it is the personality trait neuroticism (N) that interacts with the 5-HTTLPR genotype to cause depression. They demonstrate that if N is included in the model, the interaction between SLE and 5-HTTLPR becomes non-significant in their sample of female twins. However, from the graphic presentation of their data (Figure 2, p. 992),29 it is clear that while N explains most of the main effect of SLEs, the interaction between SLE and 5-HTTLPR (line slope) changes very little. This reflects the strong collinearity between SLE and neuroticism, consistent with previous reports.146 Given the very strong prospective association between neuroticism and depression,62 it is not surprising that the relatively weak 5HTTLPR × SLE interaction became non-significant when neuroticism was included in the model. It appears that in this particular study, neuroticism was a better measure of individual–environment incongruence than the self-report measure of SLE. Neuroticism and depression are overlapping concepts determined by common genetic factors.62 Furthermore, neuroticism is associated with both SLE144 and with the 5-HTTLPR.147, 148, 149 The genetic contribution to N is substantial150 and unlike SLE, the genetic and environmental components cannot be separated in its measurement. Therefore if environmental adversity is replaced with N, the G × E interaction would risk becoming a covert GxG one. This would not further our understanding of the aetiology of depression. Instead, we suggest focussing on independent and objectively recorded SLE to purify the ‘E’ in future G × E research.

Positive and negative environment

Most of the G × E studies focussed on the depressogenic effect of negative environmental factors. However, the two studies that included positive components of the environment, suggest that individuals with the s 5-HTTLPR allele are not only vulnerable to negative experiences, but also derive more benefit from positive events and social support.30, 35 Thus it appears that this genotype does not convey ‘vulnerability’ but rather renders its carriers more sensitive and reactive to life circumstances in general. This would explain the lack of direct association between depression and 5-HTTLPR polymorphism.151

The breath and depth of the phenotype

Comorbidity, depression, substance abuse and suicide

Most known environmental and genetic risk factors are not diagnosis-specific. The studies summarised above reported that 5-HTTLPR genotype moderates the effect of environmental adversity on the onset of diagnosed depression,11, 15, 32, 35, 37 on a continuous measure of depressive symptoms,29, 31, 34, 40, 41, 42 on severity of depression within a group of diagnosed patients36 and on suicidality.11, 47 Additional recent studies have found similar relationship for alcohol and drug use43, 59, 152 and for violent criminal behaviour.153 Evidence from animal studies also supports the 5-HTTLPR G × E in the genesis of alcohol dependence.8, 154 On the other hand, there appears to be some degree of specificity as no G × E was identified for generalised anxiety disorder or anxiety symptoms.15, 34

While 5-HTTLPR genotype is not reliably directly associated with depression,151 the more severe and multiple adverse outcomes including violent suicide,155, 156 suicide attempts among alcohol-dependent subjects157 and comorbid alcohol dependence with depression158, 159 are directly associated with 5-HTTLPR s allele loading. Although a meta-analysis reported a weak association between 5-HTTLPR s allele and alcoholism per se,158 this association was driven by samples with comorbid mental health problems.

Depression, substance dependence and suicidality are likely to be components of a phenotype caused by interaction between 5-HTTLPR s allele and environmental adversity. It is possible that other aspects of this phenotype will be discovered, for example general tendency to self-destructive behaviour. The multiple related outcomes of the 5-HTTLPR G × E pathogenic pathway could be due to either of the following mechanisms: (1) multiple effects of the same gene and environment (G × E pleiotropy); (2) subsequent elements on one causal pathway: once depression or substance abuse is present in an individual carrying an s allele, this condition may act as a stressor, contributing to the development of comorbidity; (3) may be markers of a latent underlying feature, which is more directly linked with the genotype, that is an intermediate phenotype or ‘endophenotype’.160

Psychological and biological endophenotypes

A discovery of intermediate phenotype that is more heritable or more strongly associate with a specific polymorphism than a diagnosed psychiatric disorder, would further our understanding of the mechanism of the observed G × E. An obvious candidate is neuroticism, which in its essence is an enduring trait of exaggerated negative emotional reactivity to events. Neuroticism is highly heritable.150 An association of neuroticism with 5-HTTLPR short allele has been reported and withstood the test of meta-analyses.72, 147, 148, 149 However, the small effect size of this association, dependence on measure of neuroticism used and the uncertainty of recessivity or dominance of the s allele effect suggest that the present concept of ‘neuroticism’ does not fit the putative trait associated with the 5-HTTLPR ss genotype. Facets of neuroticism may be explored as well as other psychological traits related to the reactivity to events, including ruminative response style,161 instability of self-esteem,162 or experiential avoidance.163 Paradigms measuring behaviours under specified circumstances may overcome the limitations of self-report questionnaires and prove to be valid measures of intermediate phenotypes.164, 165, 166, 167, 168 For example, it has been demonstrated that 5-HTTLPR s allele carriers have more attentional bias towards anxiety-related stimuli compared to ll homozygotes in the dot-probe task.168

A complementary approach will be the identification of biological endophenotypes.169 Given that the 5-HTTLPR polymorphism is in the regulatory promoter region of the serotonin transporter gene, it is likely that dynamic endophenotypes, concerning response to a challenge or a stressor will be of greater value in disentangling the mechanism of this G × E. Promising examples include response to tryptophan depletion,90, 93, 94 HPA activation by stress and response to corticosteroids,71 and clinical response to serotonin reuptake inhibitors.111 Exploring the interplay between psychological and biological intermediate phenotypes is one way forward to disentangle and characterise the G × E.

Analysis

The reviewed studies used varied statistical approaches including logistic regression’11, 32, 35, 40, 45 linear regression,11, 29, 43 survival analysis with proportional hazards regression15 and generalised estimated equations.31

A simulation study has shown that logistic regression analysis with dichotomised categorical outcomes (for examples diagnosis) can spuriously suggest an interaction where only additive effects of genes and environment on liability to the categorical outcome are present.170 The proneness to artefacts is further increased if selected samples of individuals on the extremes of liability are analysed. The reported series of simulations suggest that linear approach with random samples is the most robust approach. However, it is advised to probe the specificity of any hypothesised G × E interactions by including other variables with main effect but without expected interactions.170 In view of these data, it would be advisable to focus on continuous outcome measures in random samples and to include ‘control’ environmental risk factors without an expected G × E. As each individual study is bound to be limited by some features of its design or analysis, a meta-analytical approach will be valuable in testing the G × E. Comprehensive reporting of descriptive variables by gender, age and genotype groups should be encouraged to facilitate meta-analysis.

Interpreting the results

Gene-disorder association, G × G or G × E interaction?

While the G × E concept is proving useful in describing the interplay of risk factors in varied populations, it is important to realise that it is not independent of the distribution of genetic and environmental factors in a specific sample. If a great majority of a sample is exposed to the environmental factor, a main effect of gene will be more likely to be identified than a G × E. Conversely, if the environmental exposure is rare, no main effect of gene or G × E will be identified. Thus, the variability in E may determine the difference between G × E, main G or no effect. Therefore it is essential to demonstrate that a measure of environmental risk factor is reliable, valid and has a reasonable range of distribution in a specific sample where G × E interaction is to be tested. In the G × E studies published so far, the lack of strong main E effect appears to be associated with negative findings of G × E. This indicates low variability or reliability of the E measure, which invalidates the G × E test.

Another conceptual issue is the genetic control of exposure to environment, which has been discussed above. If there is a strong genetic component in the measure of E, the apparent G × E may mask a G × G epistatic effect. As the implications of G × E and G × G for the prevention of mental illness are radically different, it is crucial to separate the two. This can be done by distilling a pure E factor using measures of independent or objectively recorded SLE or by controlling for the heritable factor in statistical modelling.

The paradox of no main effect

Large proportion of general population experience stressful life events and other forms of environmental adversity. Carriers of an allele that confers vulnerability to develop depression in response to adversity should overall be more likely to have depression due to a substantial proportion of them encountering adversity. However, this does not appear to be the case. While there have been positive association studies of 5-HTTLPR short allele with depression, these have generally not been replicated171 and a recent meta-analysis showed no evidence of association.151 Similarly, only one of the studies attempting to replicate the findings by Caspi et al. found a direct association between depression and genotype.37 Studies with adolescent samples have indicated a weak inverse main effect, that is depression associated with 5-HTTLPR long allele.40, 42

This pattern of findings suggests that the ‘vulnerability’ in face of adversity is balanced by a protective mechanism associated with the same genotype. The nature of this protective effect is unclear. One answer comes from childhood studies showing that the same genotype is associated with vulnerability to adversity and with benefit from positive environmental factors (social support).31, 152 These results suggest that the 5-HTTLPR short allele confers a general sensitivity to both positive and negative aspects of environment. It remains to be established whether this sensitivity extends to adolescence and adulthood and what are the underlying neural and psychological mechanisms.

Conclusions

The genetic mediation by 5-HTTLPR of vulnerability to adverse environment identified by Caspi et al. (2003) appears plausible as it is supported by quantitative population genetics studies,5 neurophysiological investigations102 and experimental studies in animals.26 The majority of replication studies have provided results consistent with such effect (Table 1). Potential explanations for some discrepant findings are sample age composition, selected samples and use of unreliable measures of the environment. There are important unresolved issues including the effect of sl heterozygosity, the role of the rs25531 SNP, and the (in)dependence of ‘environment’ on personality traits and genotype. It is also unclear whether adolescent boys are protected from the pathogenic G × E or whether the contemporary methods are inadequate to measure exposures and outcomes relevant to this group.

Future directions

To exploit the potential of G × E, the unresolved issues highlighted in the previous paragraphs will have to be addressed in carefully designed studies. In the following paragraph and in Table 2, we will attempt to distil the information from the reviewed literature into practical recommendations for future research.

Table 2 Potential sources of inconsistent findings in published literature and how these could be addressed in future research

The sample selection appears to be of critical importance. Large random representative samples are clearly preferable. Correction methods, such as the genomic control, are available and should be used to control for non-random selection or genetic admixture.172 As gender and age groups differ in the vulnerability to environmental adversity, prevalence of relevant outcomes and possibly the G × E mechanism itself, it would be beneficial to routinely use and report analysis stratified by age and gender. Specific groups, such as adolescent boys may merit separate investigation.

The genotype definition has undergone some development since the first 5-HTTLPR G × E studies. As there is no consensus on allele dominance and modification by the rs25531 polymorphism, results should be reported separately for each genotype; fit of alternative models of the genetic effect should be tested in each sample and subsequently in meta-analyses. Epistasis with genes with strong evidence-based rationale, such as BDNF, has a potential to further refine the concept and indicate mechanisms underlying the observed G × E.

The chief advantage of the G × E concept is that two independently determined elements are required and elimination of their co-occurrence will prevent the adverse outcome. This concept requires a pure measure of environment, independent of genetic effects. While a complete independence may not be possible, it can be maximised using established methods of the life-event research. Follow-up after an objectively recorded adverse event in a natural experiment study is a promising avenue not yet exploited in the G × E research.

It is possible that focus on single diagnosis decreases the power to detect general aetiological pathways relevant to a number of adverse outcomes. We suggest that simultaneous exploration of multiple adverse outcomes including depression, substance use, risk-taking and self-destructive behaviours may help to resolve inconsistencies and broaden the G × E concept to groups that are currently poorly understood.

Finally, the aim of G × E researchers is to describe the causal pathway from the gene to the outcome. The use of biological and behavioural intermediate phenotypes is likely to bridge the gap.

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Acknowledgements

We would like to thank Avshalom Caspi and Terrie Moffitt for their helpful comments on previous versions of the manuscript.

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Correspondence to R Uher.

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Uher, R., McGuffin, P. 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 (2008). https://doi.org/10.1038/sj.mp.4002067

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Keywords

  • gene–environment interaction
  • depression
  • substance abuse
  • serotonin transporter
  • stressful life events

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