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. 2011 May 12;2(1):103–109. doi: 10.1016/j.dcn.2011.05.001

Influence of the OPRM1 gene polymorphism upon children's degree of withdrawal and brain activation in response to facial expressions*

Eleonora Bertoletti a,*, Annalisa Zanoni a, Roberto Giorda b, Marco Battaglia a
PMCID: PMC6987671  PMID: 22682732

Highlights

* The OPRM1-A118G polymorphism is associated with higher levels of social withdrawal. * The OPRM1-A118G polymorphism is associated with enhanced N170 to emotional faces. * Social withdrawal predicts larger N170 amplitude only for the anger expression.

Abbreviation: EoE, expressions of emotions

Keywords: OPRM1, Social rejection, Social withdrawal, Event-related potentials, Emotional expressions

Abstract

Genetic variation of the A118G polymorphism of the μ-opioid receptor gene (OPRM1) predicts individual sensitivity to social rejection and fMRI activation during simulated social rejection in adults, while data on these relationships during childhood are lacking. We investigated whether this polymorphism predicts childhood withdrawal – a predictor of sensitivity to social rejection –, and the face-specific N170 event-related waveform in response to facial expressions. Among facial expressions, ‘anger’ was expected to be particularly evocative, as it communicates social rejection.

Forty-nine children aged 8–10 years were characterised for their OPRM1 genotype, their score at the Withdrawn Scale of the Child Behavior Checklist (CBCL), and for N170 latencies and amplitudes recorded during a task of implicit processing of happy, neutral, and angry expressions of other children. Children carrying the OPRM1-G allele had higher CBCL Withdrawn scores and enhanced N170 amplitudes in response to facial expressions. Multiple linear regressions showed that the Withdrawn scale score predicts larger N170 amplitudes at the Pz and C4 electrodes, only for the anger expression.

Children who carry one or two copies of the OPRM1 G-allele are more likely to manifest withdrawn behaviours, and differ for electrophysiological responses to the early phases of processing affective stimuli.

1. Introduction

In pronouncedly social species like man and other primates social rejection can constitute an especially punitive condition (Slavich et al., 2010), but individuals can differ widely for the degree of sensitivity to social rejection during development (Sebastian et al., 2010). Sensitivity to social rejection can be predicted in part by psychological dispositional traits, such as interpersonal sensitivity in adulthood (Mehrabian, 1994), and social withdrawal in childhood. Social withdrawal has been defined as “the consistent display across situations and over time of all forms of solitary behavior when encountering familiar and/or unfamiliar peers” (Rubin and Asendorpf, 1993). Compared to non-withdrawn peers, children who are socially withdrawn appear more shy, refrain from initiating contact with peers, are less talkative and display higher rates of solitary onlooking behavior (Gazelle, 2010). Even if social withdrawal is not a pathological dimension per se, the most withdrawn children are at heightened risk for specific psychopathological outcomes such as Social Phobia (Aschenbrand et al., 2005, Gazelle, 2010). Socially withdrawn children are also especially sensitive to rejection from peers and more likely than their more sociable age-mates to experience it (Nelson et al., 2005): significant correlations have been found between social withdrawal and the frequency of rejections by peers in a sample of children aged 3–5 years (Wood et al., 2002) and in students aged 17–22 years (Molden et al., 2009).

Research has recently begun to reveal similarities between the neural processes underlying social rejection and those underlying physical pain, which led to conceptualize the experience of social rejection as a form of psychologically painful experience (Eisenberger and Lieberman, 2004, Kross et al., 2011). Particularly, greater activations of the dorsal anterior cingulate cortex (dACC) and the right ventral prefrontal cortex (both involved in the perception of physical pain) have been found associated with the experience of social exclusion (Eisenberger et al., 2003) and with higher sensitivity to peer rejection (Masten et al., 2009).

The search for common biological and genetic underpinnings shared by physically and psychologically painful experiences also led to investigate the role of the μ-opioid system. This is reasonable, given the effectiveness of opioids in alleviating both physical pain and separation distress (Kalin et al., 1988, Nelson and Panksepp, 1998) in several species. The fewer ultrasonic vocalizations emitted by μ-opioid receptor knockout mouse pups during maternal separation (Moles et al., 2004) also suggest a role for the μ-opioid system in infant–mother attachment mechanisms. Some human studies are consistent with the hypothesis of an involvement of the μ-opioid system in modulating painful psychological experiences related to loss and rejection. During a task of recall of social loss experiences, reduced μ-opioid cortical and subcortical neurotransmission was associated with ratings of negative affectivity (Zubieta et al., 2003), while a PET study found an association between lower μ-opioid receptor availability and increased cortical activation during the presentation of emotional pictures (Liberzon et al., 2002).

Further investigation of the μ-opioid neurotransmission led to focus on the μ-opioid receptor gene (OPRM1). A single nucleotide polymorphism, the A118G, within the OPRM1 leads to an amino acid change (N40D) in the μ-opioid receptor. Receptors encoded by the G allele variant were initially reported to have higher affinity for endogenous opioids, compared to those encoded by the A allele (Bond et al., 1998), but subsequent research failed to replicate this finding (Befort et al., 2001, Beyer et al., 2004), and further studies found an association between the G allele and diminished μ-opioid receptor function (Zhang et al., 2005). Although more data are needed to elucidate the precise functional significance of the G allele (Miranda et al., 2010), available evidence indicates that the G variant is associated with enhanced sensitivity to pain perception, both of physical (Sia et al., 2008), and possibly psychological, valence. A recent fMRI study (Way et al., 2009) examined the association between this polymorphism, dispositional sensitivity to social rejection, and neural responses to a social rejection paradigm in adults. Results showed that during simulated social exclusion subjects carrying the G allele were significantly more sensitive to rejection and showed a greater activity in the dACC and anterior insula, compared to A homozygotes. Moreover, dispositional sensitivity to rejection was positively correlated with activity in the dACC (Way et al., 2009). In infant rhesus monkeys, the corresponding OPRM1 C77G polymorphism influences attachment behavior; specifically, G-allele rhesus carriers show persistently high levels of distress vocalization after repeated maternal separation, and are less likely to interact with other group members (Barr et al., 2008).

In addition to social exclusion paradigms, individual sensitivity to social rejection can be investigated by the reactivity to facial expressions of emotions (EoE). In an fMRI study examining the neural responses to video clips of threatening EoE, individuals who scored higher on a measure of rejection sensitivity showed greater dACC and prefrontal activity in response to facial expressions of disapproval (Burklund et al., 2007). EoE constitute primary, non-verbal social–emotional stimuli whose social meaning starts to be processed by the brain within few hundred milliseconds from presentation. This makes EoE especially suitable for investigation with time-sensitive techniques, such as cerebral visual event-related potentials (ERP). Several studies found that the N170 early negative component – which occurs around 150–200 ms post-stimulus onset and can be observed in humans from the age of 4 to 5 years (Batty and Taylor, 2006) – is a ‘face-specific’ waveform, as it is evoked by human faces but not, or to a much lesser extent, by other types of visual stimuli (Itier and Taylor, 2004). Although some earlier studies reported the N170 as being unaffected by the type of expression (Eimer and Holmes, 2002, Krolak-Salmon et al., 2001), several recent studies report an emotional modulation for this component (Batty and Taylor, 2006, Blau et al., 2007, Caharel et al., 2005, Leleu et al., 2010). Greater N170 amplitudes are associated with the EoE of anger (Wieser et al., 2010) and disgust (Caharel et al., 2005) which convey signals of social rejection, compared to neutral/positive EoE. Moreover, there is growing evidence that early ERP components in EoE processing can be modulated by individual differences in temperamental dispositions towards anxiety in children (Lewis et al., 2007) and social anxiety in adults (Muhlberger et al., 2009). Two studies found Social Anxiety Disorder (Kolassa and Miltner, 2006) and situational social anxiety (Wieser et al., 2010) associated with larger N170 amplitudes in response to angry, compared to happy and neutral, EoE in adults.

Based on these findings, we explored the possible relationships between the A118G polymorphism, individual differences in the degree of social withdrawal – a correlate of social rejection –, and ERP activity in response to facial EoE, in a sample of general population children aged 8–10 years. We hypothesized an association between the G allele, higher degree of social withdrawal, and greater cortical activation – as indexed by an enhanced N170 amplitude – in response to the rejection expression of anger. We also expected that social withdrawal would predict the N170 amplitude in the same direction.

2. Materials and methods

2.1. Subjects

This study is part of a project on the relationships that tie some polymorphic genes, neural responses to social–emotional stimuli consisting of facial EoE, and behavioural/temperamental traits in a sample of general population children. The procedures of this study were accepted by the Ethical Committees of the participating institutions.

Initially, 149 schoolchildren were characterised by behavioural/psychological variables and by their ability to discriminate other children's facial EoE (Battaglia et al., 2004). In the second stage, 49 children (23 girls and 26 boys) from the original sample agreed to take part in the electrophysiological evaluation, after parental written informed consent (Battaglia et al., 2005).

2.2. ERP and behavioural assessment protocols

As described previously (Battaglia et al., 2004, Battaglia et al., 2005, Battaglia et al., 2007), since third- and fourth-grade schoolchildren spend most of their time among other children, we chose to use standardized faces of other children of similar age (two models, a boy and a girl, aged 8–9 years), instead of adults. Stimuli consisted of 6 black-and-white pictures standardized for size, contrast and luminosity, displaying three emotions: joy, anger and a neutral expression, included to respectively reflect the conditions of acceptance-prosocialization, rejection-hostility and neutrality that characterize social interactions in childhood.

Children were told that this was a video game in which they would get a gift if they carefully followed the instructions and performed well. The task was divided into two trials, separated by a short break in order to reduce the risk for attention decline induced by stimulus repetition.

On each trial, children were first presented with a child's face (total time on screen, 1300 ms) and were instructed to ‘watch it carefully until a blue circle appeared superimposed around the center of the picture’. As soon as the blue circle appeared (700 ms after the appearance of the stimulus), they had to click on a mouse. Thus, the ERPs relevant to this study were all generated before the motor task, which was merely set up to stimulate children's participation and attention. The monitor screen remained dark between trials for periods that varied randomly from 1200 to 1600 ms. Stimuli were presented in a sequence that alternated male and female pictures and that avoided close repetition of the same expression. To simulate a real video game and sustain children's participation, screen pictures with increasing scoring values appeared about every 6 pictures. Each stimulus was presented 20 times to ensure sufficient ERP acquisition (total, 120 presentations in a complete session).

Every child was exposed to a pre-experiment trial of 6 pictures not belonging to the same set used for the experiment to make sure she or he understood the procedure well. Each child received an educational gift of value equivalent to €30.

Electroencephalographic activity was recorded at sites Fz, C3, Cz, C4 and Pz of the 10–20 system with the use of silver–silver chloride electrodes referred to linked mastoids with an amplifier (Neuroscan SynAmp, Neuroscan Labs, Sterling, VA, USA), with head preamplified gain 150 and acquisition software (SCAN, version 4.2, Neuroscan Labs). The ground electrode was attached to the forehead. Electro-oculographic activity was recorded from electrodes placed above and below the right eye. Electrode impedance was maintained below 5 kΩ.

The electroencephalogram and electro-oculogram were amplified (gain 500), analogically band-pass filtered (1–30 Hz), digitized and acquired at a1000-Hz sampling rate. Electroencephalogram and electro-oculogram epochs between −50 and 1300 ms from the stimulus onset were obtained by means of different trigger codes for each image, allowing for later off-line artifact rejection, sorting and digital averaging with the Neuroscan EDIT software (Neuroscan Labs). All epochs from all electrodes were rejected if affected by artifacts (greater than +65 μV or less than −65 μV between −50 and 700 ms). The ERP averages were constructed from artifact-free epochs for each trigger code and for each electrode. Amplitude was measured in relation to the baseline mean voltage level preceding the onset of a waveform, and latency was defined as the time that occurred between the appearance of the stimulus (set at time 0) and the waveform's peak.

After the ERP recording, all mothers and children were interviewed individually with the Italian version of the Schedule for Affective Disorders and Schizophrenia for School-age Children (K-SADS) interview to collect the children's lifetime DSM-IV symptoms of several childhood psychiatric disorders.

To assess children's degree of social withdrawal, we used the Withdrawn scale of the Child Behavior Checklist (CBCL) 4–18 (Achenbach and Rescorla, 2001), filled in by mothers. Parental occupation was employed to calculate the families’ socioeconomic status (SES) on the basis of the Hollingshead scale (Hollingshead, 1975).

2.3. DNA extraction and genotyping

Genomic DNA was extracted from mouthwash samples collected in 4% sucrose by means of a reagent for isolation of genomic DNA (DNAzol Genomic DNA Isolation Reagent; Molecular Research Center Inc., Cincinnati, Ohio).

The entire exon 1 of the OPRM1 gene was amplified with primers OPRM1-E1F (CCT TCC AGC CTC CGA ATC) and OPRM1-E1R (CCC AGT TTA CCT CCC CTC TT) using standard protocols. The resulting 707-bp fragment was sequenced on a 3130xl Genetic Analyzer (Applied Biosystems, Monza, Italy) and the OPRM1 A118G (rs1799971) polymorphism and additional rare polymorphisms rs6912029, rs41292890, and rs9282818 were scored.

2.4. Data analyses

2.4.1. Preliminary data analyses

Previous analyses (Battaglia et al., 2007) of the waveforms obtained in the facial expression task showed an early negative component occurring after stimulus presentation at a mean latency of 136, 153, 152, 154 and 159 ms, respectively for Fz, Cz, C4, C3 and Pz electrodes, compatible with those reported by studies of N170 elicitation by facial stimuli in childhood (Taylor et al., 2004) and adulthood (Eimer and Holmes, 2002). Thus, these waveforms were putatively identified as N170. According to repeated measures analysis of variance (ANOVA), the mean N170 amplitude (mean of joy, neutral and anger expressions) differed significantly across the five electrodes (F(4,96) = 47.14, p = 0.00001): the mean amplitude at Cz was significantly larger than those at Fz and Pz (both post hoc Tukey HSD p = 0.00012) and larger, albeit not significantly, than those at C3 and C4.

Similarly, repeated measures ANOVA had shown significant differences in the N170 mean latency (mean of joy, neutral and anger expressions) across the five electrodes (F(4,96) = 19.34, p = 0.00001), with latencies at central electrodes Cz, C3 and C4 not significantly different from each other, but significantly different from both Fz and Pz latencies (post hoc Tukey HSD comparisons of Cz, C3 and C4 vs. Fz and Pz p range = 0.00012–0.0022).

Previous analyses in this sample of children had also shown no evidence of significant habituation effects on either N170 amplitude or latency, and no evidence of the effect of sex and age, alone or in interaction, on either N170 amplitude or latency (Battaglia et al., 2007). According to these results, subjects were pooled in all subsequent analyses, regardless of age or sex.

2.4.2. Statistical analyses

The general effect of children's sex on the CBCL Withdrawn scale score was preliminarily investigated by analysis of covariance (ANCOVA) with age as a covariate.

The genotypic frequencies of the A118G polymorphism were compared by chi-square test to examine possible differences in the genotypes’ distribution according to age, sex and SES of participants.

For the behavioural and ERP data analyses, similarly to previous studies on the OPRM1 gene in humans (Way et al., 2009) and primates (Barr et al., 2008), and in the light of the small number of G homozygotes in our sample (n = 2), we pooled G homozygotes and A/G subjects together into a “G-carrier” group, to be contrasted to A homozygotes.

To test the hypothesis that the A118G polymorphism is associated with an higher degree of social withdrawal, we compared by t-test the scores of G-carriers vs. A homozygotes at the CBCL Withdrawn scale.

While we had a priori identified the CBCL Withdrawn scale as a reasonable measure to index social withdrawal which could be predicted by the OPRM1 genotype (G-carrier vs. A homozygote status), we also wanted to assess the degree of specificity of the relationship of the CBCL Withdrawn scale to the OPRM1 gene. To do so, we undertook a backward logistic regression where the genotype was the dependent variable and the three CBCL Internalizing subscales (Withdrawal, Somatic Complaints and Anxiety–Depression) were the predictors. Similarly, we compared by t-test the scores of the two genotypic groups at the CBCL Externalizing scale, which is factorially and behaviourally independent from the Internalizing scales (Achenbach and Rescorla, 2001), and maps externalizing behaviours such as aggressive and delinquent behaviours.

To analyze the effect of the A118G polymorphism upon the N170 parameters, we performed, for each of the 5 electrodes, two separate repeated measures ANOVAs (one for amplitude and one for latency) with genotype (G-carriers vs. A homozygotes) as the factor, the N170 amplitude or latency as the dependent variables, and the expressions (joy/neutrality/anger) as the repeated-measures factor.

In the light of the small number of G-carriers compared to A homozygotes subjects in our sample, we performed power analyses for all the above described analyses.

To examine if the degree of social withdrawal predicts the specific N170 response to the rejection expression of anger, we performed a series of multiple linear regressions with the CBCL Withdrawn scale score as the independent variable, and the N170 parameters (amplitudes or latencies) obtained at the 5 electrodes for joy/neutrality/anger as the dependent variables.

3. Results

As can be expected in a general population sample, very few children met the criteria for psychiatric disorders. According to the K-SADS interview, the frequencies of the diagnoses in the 49 participating children were: 4 for Social Phobia, 3 for Specific Phobia, 2 for Separation Anxiety Disorders and 1 for Generalized Anxiety Disorder. No children met the criteria for a diagnosis of Depression, Panic Disorder, Attention Deficit/Hyperactivity Disorder, Obsessive–Compulsive Disorder, Conduct Disorder, Oppositional Disorder and Tic Disorder. There were no relationships between any of these diagnoses and OPRM1 genotype or ERP N170.

An ANCOVA showed that the CBCL Withdrawn scale score did not differ significantly in boys and girls (F(1,46) = 0.64, p = 0.43), without significant effects of age as a covariate (F(1,46) = 2.87, p = 0.10).

Genetic data were available for 47 of the 49 children. The allelic frequencies were: 80 (85%) for the A allele and 14 (15%) for the G allele, and the genotypic frequencies were 35 A/A (75%), 10 A/G (21%), and 2 G/G (4%), which does not significantly depart from the frequencies observed in the European population (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs1799971).

According to chi-square analyses, there were no differences in the genotypes’ distribution owing to age (χ2(4) = 3.95, p = 0.41), sex (χ2(2) = 0.01, p = 0.99) or SES (χ2(4) = 2.44, p = 0.66) of participants (Table 1).

Table 1.

Demographic features across genotypes in children.

OPRM1 genotype
AA (n = 35) AG (n = 10) GG (n = 2)
Age (mean ± SD) 8.74 ± 0.66 9.00 ± 0.82 9.50 ± 0.71
Sex, N (%) boys 18 (51.4) 5 (50) 1 (50)
SES, N
 Lower 6 0 0
 Middle 17 6 1
 Upper 12 4 1
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The t-test showed that the presence of one or two copies of the G allele was associated with higher scores at the CBCL Withdrawn scale; the scores at the CBCL Externalizing scale, on the contrary, did not differ significantly between the two genotypic groups (Table 2).

Table 2.

Influence of the OPRM1 genotype on CBCL scales and ERP N170 amplitude.

OPRM1 genotype
A homozygotes G-carriers
CBCL withdrawn, mean ± SD (n) 2.37 ± 2.16 (35) 4.50 ± 3.61a (12)
CBCL externalizing, mean ± SD (n) 9.09 ± 5.38 (35) 8.25 ± 5.82b (12)
N170 amplitude (μV) Pz (n) −6.62 ± 2.93 (26) −8.98 ± 3.19c (10)
Cz (n) −10.57 ± 3.27 (33) −13.94 ± 3.20d (10)
C3 (n) −9.36 ± 3.18 (30) −12.53 ± 1.58e (8)
C4 (n) −9.40 ± 2.98 (28) −12.90 ± 2.25f (9)
Fz (n) −4.24 ± 1.85 (27) −4.86 ± 0.8g (8)
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a

t(45) = −2.46, p = 0.02.

b

t(45) = 0.46, p = 0.65.

Repeated measures ANOVA:

c

Main effect of OPRM1 genotype: F(1,34) = 4.50, p = 0.04. Main effect of expression: F(2,68) = 0.79, p = 0.46.

d

Main effect of OPRM1 genotype: F(1,41) = 8.22, p = 0.01. Main effect of expression: F(2,82) = 0.48, p = 0.62.

e

Main effect of OPRM1 genotype: F(1,36) = 7.39, p = 0.01. Main effect of expression: F(2,72) = 0.17, p = 0.84.

f

Main effect of OPRM1 genotype: F(1,35) = 10.37, p = 0.003. Main effect of expression: F(2,70) = 0.71, p = 0.49.

g

Main effect of OPRM1 genotype: F(1,33) = 0.85, p = 0.36. Main effect of expression: F(2,66) = 0.48, p = 0.62.

The results of the backward logistic regression showed that only CBCL Withdrawal maintained a significant relationship to the OPRM1 genotype (B = 0.28, S.E. = 0.13, Wald = 4.60, df = 1, p = 0.03), while the other two subscales were removed from the final solution, due to non-significant contribution (p = 0.66 and 0.25 for Anxiety–Depression and Somatic Complaints respectively).

By repeated measures ANOVAs, we found a significant main effect of the OPRM1 genotype on N170 amplitude at Pz, Cz, C3 and C4 electrodes, always in the direction of an increased response among G-carriers, with no significant effect of expression as repeated-measures factor and no interaction at any of the 5 electrodes (Table 2).

Repeated measures ANOVAs conducted on N170 latency showed no effect of genotype and no effect of expression, alone or in interaction, on this parameter at any of the 5 electrodes (data available from authors upon request).

Results of power analyses for these analyses varied between 0.52 and 0.92.

The multiple linear regressions of the CBCL Withdrawn scale score upon the three N170 amplitudes for joy, neutrality and anger expressions in the whole sample, irrespective of the OPRM1 genotype, showed a significant effect in the direction of larger N170 amplitudes for ‘anger’ at Pz (beta = −0.35, adjusted R2 = 0.10, p = 0.03) and C4 (beta = −0.50, adjusted R2 = 0.23, p = 0.001) electrodes, but no effect for ‘joy’ or ‘neutrality’.

A regression where the genotypes (G-carriers and A-homozygotes) and CBCL Withdrawal scores were included as predictors of N170 amplitude in response to the ‘anger’ expression, however, yielded beta = −0.29, adjusted R2 = 0.09, p = 0.08, with an observed power value of 0.6.

Fig. 1 shows a scatterplot of the N170 amplitude in response to ‘anger’ by the degree of Withdrawal in the whole sample, with subjects belonging to the two genotypic groups (G-carriers and A homozygotes) identified by different symbols. While a regression of the degree of Withdrawal on N170 amplitude in response to ‘anger’ yielded a significant result when it was confined to G-carriers (N = 10, beta = −0.69, adjusted R2 = 0.40, p = 0.04), the same did not hold true among A homozygotes (N = 26, beta = −0.12, adjusted R2 = −0.02, p = 0.53).

Fig. 1.

Fig. 1

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Scatterplot of the relationship between CBCL Withdrawal scale and N170 amplitude to ‘anger’ expression at Pz electrode in the two genotypic groups.

A mediation model applied to the C4 and Pz electrodes of the relationship between the A118G polymorphism and CBCL Withdrawal mediated by the N170 amplitude in response to ‘anger’, yielded significant results (Sobel test statistics = 1.62, p = 0.05 and 1.71, p = 0.04, for C4 and Pz respectively).

Finally, the Withdrawn scores did not predict the N170 latencies for any of the three emotional expressions at any of the reference electrodes (data available from authors upon request).

4. Discussion

The aim of this study was to investigate the relationships between the A118G polymorphism of the OPRM1 gene, individual disposition towards social withdrawal – as indexed by the CBCL Withdrawn scale – and the ERP N170 amplitude in the processing of emotional facial expressions, in normally developing children aged 8–10 years.

Consistent with our hypothesis, the presence of the G allele of the OPRM1-A118G polymorphism was associated with higher scores at the CBCL Withdrawn scale. Although the precise function of the G allele has yet to be determined, previous studies have shown that it is associated with reduced interactions with peers in infant primates (Barr et al., 2008) and enhanced sensitivity to social rejection in humans (Way et al., 2009). Since sensitivity to social rejection has been found associated with social withdrawal (Nelson et al., 2005), our results with the CBCL Withdrawal scale and the G allele are consistent with currently available data, and support a role for this polymorphism in explaining part of this important, interspecies trait. The absence of an association between the OPRM1 genotype and the other CBCL Internalizing and Externalizing scores also speaks in favour of a relative specificity for the relationship between the OPRM1 G allele and social withdrawal.

In the ERP data, the presence of the G allele was associated with enhanced N170 amplitude in response to all three facial expressions of joy, neutrality, and anger. Thus, while our expectation of a selective association with the angry expression as an epitome of rejection was not confirmed, these results are broadly consistent with the involvement of the μ-opioid system in modulating brain responses to social and emotional stimuli (Liberzon et al., 2002, Way et al., 2009).

Moreover, the association between the G allele and N170 amplitudes – but not latencies – suggests that G-carriers are different from A homozygotes for the degree – but not for the timing – of cortical activation in response to social emotional stimuli.

Taken together, our behavioural and ERP data may indicate that children with the OPRM1 G allele are more avoidant of interpersonal relationships and show greater electrophysiological activation in response to the human face, possibly reflecting greater attentional bias towards peers’ social signals. Both the behavioural and the electrophysiological findings may thus point towards the attitudes of sensitivity to social rejection and withdrawal that are thought to be in part regulated by opioid neurotransmission.

Finally, in line with our hypothesis, our results show that the CBCL Withdrawn scale score predicted a larger N170 amplitude selectively for the anger expression, suggesting that children who are more withdrawn are particularly sensitive to signals of social hostility during a task of implicit processing of social emotional stimuli. This result is in agreement with recent studies that found this component modulated by the type of expression (Batty and Taylor, 2006, Blau et al., 2007, Caharel et al., 2005, Leleu et al., 2010) and it is also broadly consistent with the larger N170 amplitudes in response to angry EoE found among subjects with higher-than-average social withdrawal, such as people with Social Anxiety Disorder (Kolassa and Miltner, 2006) and situational social anxiety (Wieser et al., 2010).

A clear relationship to link the OPRM1 genotype, N170 amplitudes in response to anger, and CBCL Withdrawal simultaneously in the whole sample, is lacking here. However, when one considers the relationship between the N170 amplitudes and the CBCL Withdrawn scale divided by genotype (as depicted in Fig. 1), and the results of the related regressions made separately among G-carriers and A-homozygotes, the need for replication of our design in a larger sample becomes at issue.

Three main limitations need to be taken into account.

First, although the distribution of the OPRM1 genotype in our sample is similar to the one observed in the European population, our results may be limited by the small number of G-carriers. This resulted in comparisons based on two groups (G carriers vs. A homozygotes) instead of gene-dosage (0/1/2 ‘risk’ alleles) analyses. On the other hand, investigations of the same polymorphism in both animal (Barr et al., 2008) and man (Way et al., 2009) have been carried out according to the same dichotomy.

Second, the CBCL is a widely adopted instrument in developmental psychopathology, and it yields quantitative scores that are suitable for individual differences’ approaches. However, we employed the CBCL Withdrawn scale as the only measure to assess childhood social withdrawal. The adoption of a single scale to assess disposition towards sensitivity to rejection is common to other studies with the OPRM1 (e.g., Way et al., 2009), but the use of multiple scales/informants – including children rating themselves for behavioural dimensions – would have yielded greater psychometric reliability.

Third, the association between CBCL Withdrawn and N170 amplitude in response to angry EoE was significant at Pz and C4 electrodes only. While this may limit the breadth of our findings, most ERP studies found that the N170 has maximum amplitudes and clearest waveforms at posterior (Batty and Taylor, 2006, Caharel et al., 2005) and lateral–temporal (Taylor et al., 2004) electrodes, which support the topographic localization of our findings.

5. Conclusions

Our results show an association of the OPRM1 gene with a psychological disposition towards social withdrawal in mid childhood, and relatively specific cerebral responses during implicit processing of peers’ expressions. The presence of one or two copies of the OPRM1-G allele is associated with more pronounced social withdrawal, and with enhanced N170 amplitudes in response to emotional expressions. Moreover, the disposition towards social withdrawal predicts greater N170 amplitude only in response to the ‘anger’ expression. Even if these data need replication, they suggest that the OPRM1-A118G polymorphism, and a pattern of cortical activation in response to emotional expressions contribute to the identification of individual vulnerability for social withdrawal, which in turn can lead to social anxiety in childhood.

Acknowledgements

We thank Uberto Pozzoli PhD, and all children and parents who participated to this study.

Footnotes

*

Financial support: This study was supported in part by PRIN2006061953 Grant awarded to M. Battaglia, and an Italian Ministry of Health 2009 Strategic Research Grant, to M. Battaglia. The first author of this paper is in the San Raffaele University Developmental Psychopathology PhD Program, supported in part by the CARIPLO Foundation ‘Human Talents’ Grant for Academic Centres of Excellence in Post-Graduate Teaching (Dr. Battaglia recipient).

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