Imaging genetics: perspectives from studies of genetically driven variation in serotonin function and corticolimbic affective processing.

Advances in molecular biology and neuroimaging have provided a unique opportunity to explore the relationships between genes, brain, and behavior. In this review, we will briefly outline the rationale for studying genetic effects on brain function with neuroimaging. We will then use studies of genetically driven variation in serotonin transporter function on corticolimbic structure and function to highlight the effectiveness of this strategy to delineate biological pathways and mechanisms by which individual differences in brain function emerge and potentially bias behavior and risk for psychiatric illness. In a series of studies, a relatively frequent regulatory variant of the human serotonin transporter gene (5-HTTLPR) has been demonstrated to bias the reactivity of the amygdala to salient environmental cues. Moreover, the 5-HTTLPR affects the development of a broader corticolimbic circuit and alters the functional integration of emotional information between the amygdala and medial prefrontal cortex. In turn, corticolimbic circuit function predicts individual differences in an experimental index of temperamental anxiety and, thus, might reflect a predictive biological marker of increased risk for mood disorders associated with the 5-HTTLPR.     

Genetic variation in serotonin transporter alters resting brain function in healthy individuals.

BACKGROUND: Perfusion functional magnetic resonance imaging (fMRI) was used to investigate the effect of genetic variation of the human serotonin transporter (5-HTT) gene (5-HTTLPR, SLC6A4) on resting brain function of healthy individuals. METHODS: Twenty-six healthy subjects, half homozygous for the 5-HTTLPR short allele (s/s group) and half homozygous for the long allele (l/l group), underwent perfusion functional and structural magnetic resonance imaging during a resting state. The two genotype groups had no psychiatric illness and were similar in age, gender, and personality scores. RESULTS: Compared with the l/l group, the s/s group showed significantly increased resting cerebral blood flow (CBF) in the amygdala and decreased CBF in the ventromedial prefrontal cortex. The effect of functional modulation in these regions by 5-HTTLPR genotype cannot be accounted for by variations in brain anatomy, personality, or self-reported mood. CONCLUSIONS: The 5-HTTLPR genotype alters resting brain function in emotion-related regions in healthy individuals, including the amygdala and ventromedial prefrontal cortex. Such alterations suggest a broad role of the 5-HTT gene in brain function that may be associated with the genetic susceptibility for mood disorders such as depression.     

Threat-related amygdala functional connectivity is associated with 5-HTTLPR genotype and neuroticism

Communication between the amygdala and other brain regions critically regulates sensitivity to threat, which has been associated with risk for mood and affective disorders. The extent to which these neural pathways are genetically determined or correlate with risk-related personality measures is not fully understood. Using functional magnetic resonance imaging, we evaluated independent and interactive effects of the 5-HTTLPR genotype and neuroticism on amygdala functional connectivity during an emotional faces paradigm in 76 healthy individuals. Functional connectivity between left amygdala and medial prefrontal cortex (mPFC) and between both amygdalae and a cluster including posterior cingulate cortex, precuneus and visual cortex was significantly increased in 5-HTTLPR S′ allele carriers relative to L_AL_A individuals. Neuroticism was negatively correlated with functional connectivity between right amygdala and mPFC and visual cortex, and between both amygdalae and left lateral orbitofrontal (lOFC) and ventrolateral prefrontal cortex (vlPFC). Notably, 5-HTTLPR moderated the association between neuroticism and functional connectivity between both amygdalae and left lOFC/vlPFC, such that S′ carriers exhibited a more negative association relative to L_AL_A individuals. These findings provide novel evidence for both independent and interactive effects of 5-HTTLPR genotype and neuroticism on amygdala communication, which may mediate effects on risk for mood and affective disorders.     

The association between the 5-HTTLPR and neural correlates of fear conditioning and connectivity

Strong evidence links the 5-HTTLPR genotype to the modulation of amygdala reactivity during fear conditioning, which is considered to convey the increased vulnerability for anxiety disorders in s-allele carriers. In addition to amygdala reactivity, the 5-HTTLPR has been shown to be related to alterations in structural and effective connectivity. The aim of this study was to investigate the effects of 5-HTTLPR genotype on amygdala reactivity and effective connectivity during fear conditioning, as well as structural connectivity [as measured by diffusion tensor imaging (DTI)]. To integrate different classification strategies, we used the bi-allelic (s-allele vs l/l-allele group) as well as the tri-allelic (low-functioning vs high-functioning) classification approach. S-allele carriers showed exaggerated amygdala reactivity and elevated amygdala-insula coupling during fear conditioning (CS + > CS−) compared with the l/l-allele group. In addition, DTI analysis showed increased fractional anisotropy values in s-allele carriers within the uncinate fasciculus. Using the tri-allelic classification approach, increased amygdala reactivity and amygdala insula coupling were observed in the low-functioning compared with the high-functioning group. No significant differences between the two groups were found in structural connectivity. The present results add to the current debate on the influence of the 5-HTTLPR on brain functioning. These differences between s-allele and l/l-allele carriers may contribute to altered vulnerability for psychiatric disorders.     

The symptomatic profile of panic disorder is shaped by the 5-HTTLPR polymorphism

The short allele of a functional polymorphism (5-HTTLPR) in the serotonin transporter (5-HTT) promoter is associated with reduced serotonin transporter expression, lower in vivo 5-HTT binding, higher neuroticism and amygdala reactivity as well as facilitated fear conditioning and is a candidate variant for panic disorder. However, case–control studies have consistently failed to show an association between 5-HTTLPR and panic disorder. As psychiatric disorders are broadly defined phenotypes merging different symptoms to syndromes, they may not be very well suited for genetic association studies. An alternative approach is to measure symptoms along continuous symptom dimensions which may more appropriately reflect their biological underpinnings and may reveal subpopulations within clinical diagnostic groups. We recorded the symptomatic profile in 73 in panic disorder patients using observer-rated instruments (Panic Disorder Severity Scale, PDSS; Montgomery–Åsberg Depression Rating Scale, MADRS) and hypothesized more severe symptoms in patients carrying the 5-HTTLPR s-allele. We observed a strong association between bi- and triallelic 5-HTTLPR polymorphisms and the symptomatic profile. Carriers of the 5-HTTLPR s-allele suffered from most severe panic and depressive symptoms. Our data highlight the importance of defining appropriate phenotypes for psychiatric genetic studies and demonstrate that the 5-HTTLPR genotype may be related to the symptomatic profiles rather than to the vulnerability to develop panic disorder.     

How the serotonin transporter 5-HTTLPR polymorphism influences amygdala function: the roles of in vivo serotonin transporter expression and amygdala structure

The serotonin transporter-linked promoter region (5-HTTLPR) polymorphism of the serotonin transporter gene is associated with amygdala response during negative emotion. The aim of this study was to investigate whether this genotype effect on amygdala function is mediated by current serotonin transporter (5-HTT) levels or rather by genetically induced influences during neurodevelopment, shaping brain structure. A total of 54 healthy subjects underwent functional and structural magnetic resonance imaging, [¹¹C]DASB positron emission tomography and 5-HTTLPR genotyping to analyze the interrelationships between amygdala activation during processing of unpleasant stimuli, 5-HTTLPR genotype, amygdala volumes and 5-HTT levels in the midbrain and in other brain regions. In line with previous research, carriers of the short allele (S) showed increased amygdala activation. Path analysis demonstrated that this genotype effect was not procured by current 5-HTT availability but by amygdala structure, with smaller amygdala volumes in the S than in the LL genotype, as well as smaller volumes being associated with increased amygdala activation. Our findings stress the role of genetic effects during neurodevelopment.     

Cognitive appraisal and life stress moderate the effects of the 5-HTTLPR polymorphism on amygdala reactivity

The short allele of the serotonin-transporter-linked promoter region (5-HTTLPR) polymorphism is associated with increased amygdala activation in response to emotional stimuli. Although top-down processes may moderate this association, available evidence is conflicting, showing the genotype influence on amygdala reactivity to be either decreased or increased during emotion regulation. Because the effects of the 5-HTTLPR polymorphism on amygdala reactivity are also conditional on self-reported life stress, differences in life stress exposure may account for this apparent discrepancy. Here, we hypothesized that self-reported life stress would moderate the relationships between genotype, cognitive appraisal, and amygdala reactivity. Forty-five healthy never-depressed subjects were presented with emotional stimuli and performed two cognitive tasks: a self-referential task and an emotion-labeling task. Life-stress exposure was measured through a semistructured interview. First, there was a genotype × condition interaction in the right amygdala: short allele carriers displayed increased amygdala activation and decreased functional connectivity with the subgenual anterior cingulate cortex in self-referential processing versus emotion labeling. Second, in line with our hypothesis, there was a genotype × condition × stress interaction in bilateral amygdala the amygdala activation during self-referential processing was negatively correlated with self-reported life stress in short allele carriers and positively in individuals homozygous for the long allele, whereas an opposite pattern was observed during emotion labeling. These results confirm that the influence of the 5-HTTLPR polymorphism on amygdala reactivity is at least partially under cognitive control. Additionally, they suggest that measuring life stress exposure is a critical step when imaging genetics.     

5-HTTLPR,anxiety and gender interaction moderates right amygdala volume in healthy subjects

Genetic variants within the serotonin transporter gene (5-HTTLPR) impact the neurobiology and risk for anxiety-related behaviours. There are also gender differences in the prevalence of anxiety-related behaviours. Although numerous studies have investigated the influence of 5-HTTLPR genotype on the neural systems involved in emotional regulation, none have investigated how these effects are modulated by gender and anxiety. We investigated this issue using two complementary region of interest-based structural neuroimaging approaches (voxel-based morphometry and Freesurfer) in 138 healthy individuals categorized into ‘no anxiety’ and ‘subclinical anxiety’ groups based on the Hamilton Rating Scale for Anxiety (HAM-A). Preliminarily, using anxiety as a continuous variable, we found a significant interaction effect of genotype by gender on anxiety. Females homozygous for the Short allele showed the highest HAM-A scores and males the lowest. In addition, a three-way significant interaction among genotype, gender and anxiety category was found for the right amygdala volume. Post hoc tests revealed that homozygous females carrying the Short variant with a subclinical anxiety condition had larger volume. The reported interaction effects demonstrate that gender strongly modulates the relationship between 5-HTTLPR genotype and subclinical expression of anxiety acting on amygdala, one region of the emotional neural network specifically involved in the anxiety-like behaviours.     

Individual differences in neural correlates of fear conditioning as a function of 5-HTTLPR and stressful life events

Fear learning is a crucial process in the pathogeneses of psychiatric disorders, which highlights the need to identify specific factors contributing to interindividual variation. We hypothesized variation in the serotonin transporter gene (5-HTTLPR) and stressful life events (SLEs) to be associated with neural correlates of fear conditioning in a sample of healthy male adults (n = 47). Subjects were exposed to a differential fear conditioning paradigm after being preselected regarding 5-HTTLPR genotype and SLEs. Individual differences in brain activity as measured by functional magnetic resonance imaging (fMRI), skin conductance responses and preference ratings were assessed. We report significant variation in neural correlates of fear conditioning as a function of 5-HTTLPR genotype. Specifically, the conditioned stimulus (CS⁺) elicited elevated activity within the fear-network (amygdala, insula, thalamus, occipital cortex) in subjects carrying two copies of the 5-HTTLPR S′ allele. Moreover, our results revealed preliminary evidence for a significant gene-by-environment interaction, such as homozygous carriers of the 5-HTTLPR S′ allele with a history of SLEs demonstrated elevated reactivity to the CS⁺ in the occipital cortex and the insula. Our findings contribute to the current debate on 5-HTTLPR x SLEs interaction by investigating crucial alterations on an intermediate phenotype level which may convey an elevated vulnerability for the development of psychopathology.     

The serotonin transporter gene polymorphism (5-HTTLPR) and affective symptoms among women diagnosed with borderline personality disorder

Gene variants of the serotonin transporter have been associated with vulnerability to affective disorders. In particular, the presence of one or two copies of the short (s) allele of the 5-HTTLPR polymorphism has been associated with reduced serotonin transporter expression and function, and vulnerability to affective disorders. To test for an association between variants of the serotonin transporter gene polymorphism (5-HTTLPR) and relevant clinical features of borderline personality disorder (BPD), a psychiatric disorder with symptoms characteristic for serotonin dysfunction, 77 women with BPD were genotyped in the 5-HTTLPR polymorphism. They rated their subjective experience of borderline-specific, depressive, anxious and obsessive-compulsive symptoms, and were interviewed about lifetime incidence of suicide attempts and self-harming acts. Carriers of two s alleles of the 5-HTTLPR reported more symptoms of borderline, depression, anxiety and obsessive-compulsive behaviours, but not of suicidal and self-injury behaviour, compared to carriers of a long (l) allele. This indicates that the 5-HTTLPR ss homozygous genotype might influence serotonin function affecting susceptibility to both borderline-specific, depressive, anxious and obsessive-compulsive symptoms in BPD, and leading to a more severe symptomatology related to these clinical features. Further, this suggests that 5-HTT gene variants may not be as influential on suicidal and self-injury behaviour in BPD.     

Gene-gene effects on central processing of aversive stimuli

Emotional reactivity and regulation are fundamental to human behavior. As inter-individual behavioral variation is affected by a multitude of different genes, there is intense interest to investigate gene-gene effects. Functional sequence variation at two genes has been associated with response and resiliency to emotionally unpleasant stimuli. These genes are the catechol-O-methyltransferase gene (COMT Val158Met) and the regulatory region (5-HTTLPR) of the serotonin transporter gene. Recently, it has been proposed that 5-HTT expression is not only affected by the common S/L variant of 5-HTTLPR but also by an A to G substitution. Using functional magnetic resonance imaging, we assessed the effects of COMT Val(158)Met and both 5-HTT genotypes on brain activation by standardized affective visual stimuli (unpleasant, pleasant, and neutral) in 48 healthy subjects. Based on previous studies, the analysis of genotype effects was restricted to limbic brain areas. To determine allele-dose effects, the number of COMT Met158 alleles (i.e., lower activity of COMT) and the number of 5-HTT low expressing alleles (S and G) was correlated with the blood oxygen level-dependent (BOLD) response to pleasant or unpleasant stimuli compared to neutral stimuli. We observed an additive effect of COMT and both 5-HTT polymorphisms, accounting for 40% of the inter-individual variance in the averaged BOLD response of amygdala, hippocampal and limbic cortical regions elicited by unpleasant stimuli. Effects of 5-HTT and COMT genotypes did not affect brain processing of pleasant stimuli. These data indicate that functional brain imaging may be used to assess the interaction of multiple genes on the function of neuronal networks.     

Association and linkage of anxiety-related traits with a functional polymorphism of the serotonin transporter gene regulatory region in Israeli sibling pairs

A functional polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) has been reported to be both associated and linked to anxiety-related personality measures, although other studies have not replicated these findings. The current study examines both association and linkage of the gene to two major anxiety-related personality measures, the harm avoidance scale on the Tridimensional Personality Questionnaire and the neuroticism scale of the NEO-PI-R, in a sample of 148 Israeli subjects comprising 74 same-sex sibling pairs. We replicated the reported association between the short allele and higher scores on the TPQ harm avoidance scale (P = 0.03), including the subscale of shyness (P = 0.02), and also found association in the same direction between the short allele and the NEO-PI-R neuroticism subscales of anxiety (P = 0.03) and depression (P = 0.04). Sib-pair linkage analysis, using the regression method, further supported a role of the 5-HTTLPR in anxiety-related personality traits.     

Cumulative activation during positive and negative events and state anxiety predicts subsequent inertia of amygdala reactivity

Inertia, together with intensity and valence, is an important component of emotion. We tested whether positive and negative events generate lingering changes in subsequent brain responses to unrelated threat stimuli and investigated the impact of individual anxiety. We acquired fMRI data while participants watched positive or negative movie-clips and subsequently performed an unrelated task with fearful and neutral faces. We quantified changes in amygdala reactivity to fearful faces as a function of the valence of preceding movies and cumulative neural activity evoked during them. We demonstrate that amygdala responses to emotional movies spill over to subsequent processing of threat information in a valence-specific manner: negative movies enhance later amygdala activation whereas positive movies attenuate it. Critically, the magnitude of such changes is predicted by a measure of cumulative amygdala responses to the preceding positive or negative movies. These effects appear independent of overt attention, are regionally limited to amygdala, with no changes in functional connectivity. Finally, individuals with higher state anxiety displayed stronger modulation of amygdala reactivity by positive movies. These results suggest that intensity and valence of emotional events as well as anxiety levels promote local changes in amygdala sensitivity to threat, highlighting the importance of past experience in shaping future affective reactivity.     

Interaction between the tryptophan hydroxylase gene and the serotonin transporter gene in schizophrenia but not in bipolar or unipolar affective disorders

Increasing focus is being given to identify possible combinations of genes related to specific clinical phenotypes. In our sample of 814 patients comprising 114 with schizophrenia, 416 with bipolar affective disorder and 284 with unipolar affective disorder, we studied interactions between the tryptophan hydroxylase (TPH), the serotonin transporter (5-HTTLPR), and the dopamine receptor (DRD4) genes in relation to five major psychiatric symptomatology scores. There was significant interaction between the TPH and the 5-HTTLPR genes. With an increasing number of short (s) alleles of 5-HTTLPR, the scores for delusions, disorganization and negative symptoms were significantly decreasing among subjects having the TPH genotype AA but increasing among subjects having the TPH genotype AC, yielding the highest scores for the combinations AA x ll and AC x ss. Since high scores on just delusions, disorganization and negative symptoms but low scores on excitement and depression were found among subjects with schizophrenia, we conducted comparisons among the three diagnostic categories and controls as regards the combined TPH x 5-HTTLPR genotype distribution. Schizophrenia subjects had a significantly different distribution of the genotype combination for TPH x 5-HTTLPR as compared to 241 controls or to unipolar or bipolar subjects, and had significantly higher frequencies of AA x ll and of AC x ss. Thus, an interaction between TPH and 5-HTTLPR genes constitutes susceptibility to schizophrenia, thereby yielding apparent relationships between the major psychiatric symptomatology scores and genotype combinations in samples that are obtained by pooling schizophrenia with other diagnostic categories.     

Serotonin transporter gene polymorphism and schizoid personality traits in the patients with psychosis and psychiatrically well subjects.

BACKGROUND AND OBJECTIVES: serotonin transporter (5-HTT) gene allelic variants were shown to be associated with Neuroticism and Harm Avoidance but the results were not replicated in other studies. The current investigation was undertaken in a further attempt to study the relationship between 5-HTT polymorphism and personality traits. SUBJECTS AND METHODS: to evaluate a spectrum of personality traits, MMPI was administered to a sample including patients with affective disorders (n=114), patients with schizophrenia spectrum illnesses (n=110) and psychiatrically well controls (n=124). All groups were genotyped for VNTR-17 and functional insertion-deletion (5-HTTLPR) polymorphisms. RESULTS: an association was found between 5-HTTLPR polymorphism and scores on three MMPI scales: Psychopathic deviance, Paranoia and Schizophrenia in patients with affective disorders and S chizophrenia in normal subjects. Both affected and control individuals with 'ss' genotype exhibited lower scores on these scales. CONCLUSION: we demonstrated that functional deletion/insertion allelic variation associated with decreased expression of serotonin transporter ('s' allele or 'ss' genotype) may restrict expression of schizoid traits in normal subjects and patients with affective disorders.     

The power of sample size and homogenous sampling: association between the 5-HTTLPR serotonin transporter polymorphism and major depressive disorder.

BACKGROUND: Several lines of evidence indicate that abnormalities in the functioning of the central serotonergic system are involved in the pathogenesis of affective illness. A 44-base-pair insertion/deletion polymorphism in the 5' regulatory region of the serotonin transporter gene (5-HTTLPR), which influences expression of the serotonin transporter, has been the focus of intensive research since an initial report on an association between 5-HTTLPR and depression-related personality traits. Consistently replicated evidence for an involvement of this polymorphism in the etiology of mood disorders, particularly in major depressive disorder (MDD), remains scant. METHODS: We assessed a potential association between 5-HTTLPR and MDD, using the largest reported sample to date (466 patients, 836 control subjects). Individuals were all of German descent. Patients were systematically recruited from consecutive inpatient admissions. Control subjects were drawn from random lists of the local Census Bureau and screened for psychiatric disorders. RESULTS: The short allele of 5-HTTLPR was significantly more frequent in patients than in control subjects (45.5% vs. 39.9%; p = .006; odds ratio = 1.26). CONCLUSIONS: These results support an involvement of 5-HTTLPR in the etiology of MDD. They also demonstrate that the detection of small genetic effects requires very large and homogenous samples.     

Afraid to help: Social anxiety partially mediates the association between 5-HTTLPR triallelic genotype and prosocial behavior

There is growing evidence that the serotonin system influences prosocial behavior. We examined whether anxiety mediated the association between variation in the serotonin transporter gene regulatory region (5-HTTLPR) and prosocial behavior. We collected self-reported tendencies to avoid certain situations and history of helping others using standard instruments and buccal cells for standard 5-HTTLPR genotyping from 398 undergraduate students. Triallelic 5-HTTLPR genotype was significantly associated with prosocial behavior and the effect was partially mediated by social anxiety, such that those carrying the S′ allele reported higher levels of social avoidance and lower rates of helping others. These results are consistent with accounts of the role of serotonin on anxiety and prosocial behavior and suggest that targeted efforts to reduce social anxiety in S′ allele carriers may enhance prosocial behavior.     

Effect of a triallelic functional polymorphism of the serotonin-transporter-linked promoter region on expression of serotonin transporter in the human brain

OBJECTIVE: The authors examined effects of a triallelic functional polymorphism of the human serotonin-transporter-linked promoter region (5-HTTLPR) on in vivo expression of serotonin transporter in the brain in healthy volunteers and subjects with major depressive disorder. METHOD: Twenty-five medication-free subjects with DSM-IV major depressive disorder during a major depressive episode and 42 healthy volunteers were clinically evaluated and genotyped. Serotonin transporter binding potential (f(1)B(max)/K(d)) was determined by using positron emission tomography with the radiotracer [(11)C]McN 5652 and metabolite-corrected arterial input functions. RESULTS: There was no difference in serotonin transporter binding potential by genotype in healthy volunteers or in subjects with major depressive disorder. Allelic frequencies did not differ between subjects with major depressive disorder and healthy volunteers. CONCLUSIONS: Associations of the 5-HTTLPR polymorphism to clinical phenotypes appear to be due to developmental effects of 5-HTTLPR on expression and not due to its direct effect on serotonin transporter binding in adulthood.     

A polymorphism (5-HTTLPR) in the serotonin transporter promoter gene is associated with DSM-IV depression subtypes in seasonal affective disorder

Serotonergic mechanisms are thought to play an important role in the pathogenesis of seasonal affective disorder (SAD). The expression of the serotonin transporter (5-HTT) is regulated in part by an insertion/deletion polymorphism in the serotonin transporter gene promoter region (5-HTTLPR). The 5-HTTLPR short allele (s) has been associated with anxiety-related personality traits and depression, and one study observed an association between the 5-HTTLPR s-allele and SAD and the trait of seasonality. We genotyped 138 SAD patients and 146 healthy volunteers with low seasonality for 5-HTTLPR. No difference between patients and controls was found for genotype distribution and s-allele frequency. However, genotype distribution and allele frequencies were strongly associated with DSM-IV depression subtypes. Melancholic depression was associated with the 5-HTTLPR long (l) allele and atypical depression with the 5-HTTLPR s-allele (two-sided Fisher's exact test: genotype distribution: P=0.0038; allele frequencies: P=0.007). Our data are compatible with the hypothesis of a disease process that is not causally related to 5-HTTLPR, but involves 5-HT neurotransmission and 5-HTTLPR somewhere on its way to phenotypic disease expression.