Personality and Individual Differences 33 (2002) 227238 www.elsevier.

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Molecular genetics of shyness and aggression in preschoolers

Abstract Associations between three candidate gene polymorphisms [dopamine D4 receptor (DRD4), serotonin transporter (5-HTT), and serotonin 2C receptor (5-HT2C)] with shy and aggression-related behaviors derived from maternal report and peer play at age four were examined. We noted a signicant association of the DRD4 receptor gene with maternal report of problems with aggression at age four. Children with long versus short repeat alleles of the DRD4 gene were reported by their mothers to have signicantly more problems with aggression at age four. There were no signicant associations of the DRD4 gene with observed behavioral measures of aggression at age four. There were, in addition, no signicant associations of either of the serotonin genes with any of the maternal report and observed behavioral measures. The present study extends earlier ndings of adults to the preschool years and appears to be the rst large scale investigation to examine the molecular genetics of preschoolers temperament using behavioral and maternal report measures in normal childhood development. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Shyness; Aggression; Molecular genetics; DRD4; 5-HTT; 5-HT2C; Preschoolers

The notion that there may be a genetic basis to individual dierences in temperament is an idea that dates from the time of the early Greeks and extends to contemporary personality research. Much of the scientic support for this concept is derived from three disparate literatures (Eley & Plomin, 1997), two of which have been reliable and convincing sources for years and a third which has emerged only within the last decade. The rst literature involves studies of domesticated and laboratory animals in which there is strong evidence in support of a genetic basis to
* Corresponding author. Tel.: +1-905-525-9140; fax: +1-905-529-6225. E-mail address: [email protected] (L.A. Schmidt).
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temperament. For example, it has long been noted that inbred strains of animals can be produced that are highly fearful, defensive, aggressive, or subdued (see Plomin, DeFries, McClearn, & Rutter, 1997 for a review). A second body of work concerns ndings derived from behavioral genetics studies of human twins. Such studies often nd that monozygotic twins, raised either apart or together, appear temperamentally more similar than dizygotic twins and adopted children (see Plomin, 1989 for a review). A third source concerns recent ndings from the rapidly emerging eld of the molecular genetics of human personality (see Cloninger, Adolfsson, & Svrakic, 1996; Hamer & Copeland, 1998; Plomin & Rutter, 1998 for reviews). These studies, mostly involving human adults, have found associations between complex human behavioral traits and genes that regulate specic neurochemical systems. Overall, these three sources are beginning to converge to provide the strongest evidence to date that there may be a genetic etiology underlying some complex human personality traits. The purpose of the present study was to explore whether there may be a molecular genetic basis to individual dierences in childhood temperament as manifested through shy and aggressive social behaviors. We have been following four separate cohorts of children who have been participating in a larger longitudinal study on socio-emotional development. To date, we have found a number of reliable ndings. Individual dierences in certain patterns of infant temperament, which appear early in life, are modestly predictive of social interactive behaviors in the preschool years. For example, Fox and his colleagues (Calkins, Fox, & Marshall, 1996; Fox, Henderson, Rubin, Calkins, & Schmidt, 2001) have described two groups of infants who appear to reect distinct temperamental types. One group of infants are highly reactive and exhibit a high degree of distress to the presentation of novel auditory and visual stimuli at 4 months of age. These infants are likely to display behavioral inhibition at 14 (Calkins et al., 1996) and 24 months (Fox, Calkins, & Bell, 1994) and social reticence to unfamiliar peers at 48 months (Fox et al., 2001), and are reported by their mother as contemporaneously shy at age four (Schmidt et al., 1997). A similar set of ndings has been reported by Kagan and his colleagues (Kagan & Snidman, 1991). These infants also exhibit a distinct pattern of psychophysiological activity, including heightened fear-potentiated startle responses at age 9 months (Schmidt & Fox, 1998) and greater right frontal EEG asymmetry at 9 (Calkins et al., 1996), 14, and 48 months of age (Fox et al., 2001) and may be at risk for anxiety-related problems during the early school age years (e.g. Fox, Schmidt, Calkins, Rubin, & Coplan,1996; Hirshfeld, et al., 1992; Schmidt, Fox, Schulkin, & Gold, 1999). A second group of infants exhibit a high degree of motor arousal and positive aect to novel auditory and visual stimuli at 4 months. These infants display low levels of uncertainty, fear in response to novelty or discrepancy at 14 and 24 months of age, and are socially interactive and engaging as preschool children. Interestingly, they display, as a group, a good deal of continuity across the early childhood years. No children in this group were identied as fearful or inhibited or socially reticent at 4 years of age and few children were in the average range. Most remained high in novelty seeking and low in fear across the 4 years of the longitudinal study. This group, Fox (Fox et al., 2001) calls exuberant, also displayed consistent patterns of left frontal EEG asymmetry at 9, 14, and 48 months of age. Our study of the molecular genetics of child temperament and social behavior focuses on candidate genes involved in signaling by dopamine and serotonin, two key neurotransmitters that have previously been implicated in individual dierences in personality in adults. The dopamine D4 receptor gene (D4DR), which contains a functional repeated sequence polymorphism within

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its coding sequences, was originally studied for its role in personality traits related to novelty seeking. The initial two studies (Benjamin, Li, Patterson, Greenberg, Murphy, & Hamer, 1996; Ebstein et al., 1996) found that adults with longer versions (68 repeats) self-reported higher novelty seeking scores compared with adults with shorter versions (25 repeats) of the coding sequence polymorphism. Dopamine has been implicated as a major neuromodulator of novelty seeking because of the role it plays in inducing euphoria in humans and approach behavior in animals (Cloninger, 1987). The shorter alleles code for a receptor that is apparently more ecient in binding dopamine compared with the larger alleles (see Plomin & Rutter, 1998, for a review). Subsequently, the association between the DRD4 polymorphism and novelty seeking has been replicated in some populations (Ebstein, Nemanov, Klotz, Gritsenko, & Belmaker, 1997; Noble, Ozkaragoz, Ritchie, Zhang, Belin, & Sparkes, 1998; Ono et al., 1997; Strobel, Wehr, Michel, & Brocke, 1999; Tomitaka et al., 1999); however, several other studies have found either no association (Gelernter, Kranzler, Coccaro, Siever, New, & Mulgrew, 1997; Goldman et al., 1996; Jonsson et al., 1997; Pogue-Geile, Ferrell, Deka, Debski, & Manuck, 1998; Sander, Harms, Dufeu, Kuhn, Rommelspacher, & Schmidt, 1997; Sullivan, Field, Kennedy, Mulder, Sellman, & Joyce, 1998; Vandenbergh, Zonderman, Wang, Uhl, & Costa, 1997) or association in the wrong direction relative to the initial reports (Malhotra, Virkkunen, Rooney, Eggert, Linnoilia, & Goldman, 1996). The serotonin transporter gene was chosen as the second candidate locus for this study because of its role in anxiety-related personality traits in adults. Lesch et al. (1996) reported that adults carrying one or two copies of a short allele of a regulatory DNA sequence polymorphism in the serotonin transporter gene (5-HTTLPR) self-reported higher levels of neuroticism, anxiety, and depression compared with individuals homozygous for the long allele of this polymorphism. In vitro and in vivo expression studies have shown that the short allele leads to less gene transcription and protein production than does the long allele (Greenberg, Tolliver, Huang, Li, & Murphy, 1999; Heils et al., 1996; Lesch et al., 1996; Little et al., 1998). Moreover, serotonin has been implicated as a major neurotransmitter of anxiety and withdrawal because of its eects on regulating mood and emotional states (see Westernberg, Murphy, & Den Boer, 1996, for a review). Subsequently, the association between the serotonin transporter gene and anxiety-related states has been replicated in populations in the United States, Europe, Israel, and Japan, (e.g. see Greenberg et al., 1999, and references therein), but other studies have failed to nd any association (Ball et al., 1997; Deary, Battersby, Whiteman, Connor, Fowkes, & Harmar, 1999; Kumakiri et al., 1999). We also examined a serotonin receptor gene (5-HT2C; Lappalainen et al., 1995). Ebstein and his colleagues (Ebstein, Segman, Benjamin, Osher, Nemanov, & Belmaker, 1997) found association of a cysteine to serine coding sequence polymorphism in this gene with the personality trait of reward dependence, especially in individuals with a long DRD4 genotype, and this result has been partially replicated (Kuhn, Meyer, Nothen, Gansicke, Papassotiropoulos, & Maier, 1999). The 5-HT2C coding sequence change does not appear to alter the response of the receptor to its ligand (Lappalainen et al., 1995) and thus the mechanism of its postulated role in personality is unclear. All of the studies described earlier were based on personality data derived from self-report measures. To our knowledge, there have been no studies which have examined links between gene markers and social behavior determined by direct, systematic observation. The current study was

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an attempt to provide those data by utilizing individual dierences in observed social behavior. In particular, we collected DNA samples from children from whom we had previously examined behavioral responses during play groups with unfamiliar same-age and-gender peers as well as maternal report of behavioral problems at age four. We examined the association of the DRD4 and two 5-HT-related genes with childrens social behaviors at age four via observed behavior and maternal report. The strongest evidence that exists to date has implicated the DRD4 and 5-HT-related genes as playing some role in complex human traits. We chose to examine approach- (e.g. aggression) and avoidance- (shyness) related behaviors because these two overarching behavioral styles are analogous to those reported in the adult personality literature for which a genetic association has been reported. We predicted that children with long versus short allele repeats of the DRD4 receptor gene would be rated by their mothers as having more problems with aggression and would exhibit more observed disruptive behaviors during peer play at age four; the phenotypic expression of these behaviors at this age is conceptually linked to novelty seeking-related behaviors (i.e. an inability to regulate socially appropriate responses) in adults for which the DRD4 has been implicated. We also predicted that children with short versus long genotypes of the 5-HTT transporter gene and the Ser23 versus Cys23 allele of the 5-HT2C receptor gene would be rated by their mothers as having more problems with social adjustment and would exhibit more observed shyness during peer play at age four; shyness is highly related to anxiety and neuroticism for which the serotonin system and serotonin genes have been implicated.

1. Method 1.1. Subjects The subjects of this study were 244 preschool children (108 males, 136 females) who were tested in the laboratory within 1 month of their fourth birthday. The children were primarily Caucasian and of middle-class background. All of the parents had completed high school and a majority of the mothers and fathers were college graduates. The children were, for the most part, living with their families in or near College Park, Maryland. Original exclusion criteria were that the child had no peri- or post-natal complications, and the child had no known psychiatric or neurological problems. Of the 244 preschool children, we had complete data on 161 children. The remaining 83 children either did not complete all of the laboratory procedures, their mothers failed to complete surveys, they failed to donate cheek cells, they had relocated and were no longer in the study or they no longer wished to participate in the study at age four. The analyses reported later focus on the 161 children who had complete data at age four. 1.2. Maternal report of childhood behavioral problems Maternal perceptions of childhood behavior problems were assessed at age four with the widely used Childhood Behavior Checklist (CBCL; Achenbach & Edelbrock, 1981). The CBCL is a 113-item checklist in which parents use a three-point scale to rate how descriptive a series of behavior problems are of their child. The CBCL yields eight subscales that index a variety of

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problems that are further reduced to two broad band factors, internalizing and externalizing factors. A total problems factor is also derived that combines all of the subscales. Of particular interest to the present study were separate subscales that measured problems with social adjustment and aggression. 1.3. Peer play groups At 4 years of age, groups of four same gender children and their mothers came to the laboratory. Children and mothers waited in an area of the laboratory until all four children had arrived and all parents had been briefed and consent granted. The four children were then led into a playroom that had in it a set of attractive toys. The children were told that they were to play in this room for a while and that afterwards they would participate in a set of games. The children in each play group had never met each other. Children were given name tags that were pinned to their backs so that their names would be visible to the video camera. The sessions were unobtrusively video- and audio-taped for future coding and analysis. The quartet session comprised ve parts: Part I was free-play and lasted 15 min. During this period of time, the four children were in the rooms by themselves. Parents were in a waiting area and asked to ll out a series of questionnaires. Part II was a clean-up session which lasted 5 min. An experimenter entered the room and told the children that the free-play session had ended and that they were going to be playing a series of games. The experimenter asked the children to clean-up the toys, placing them in a larger cardboard box that had been placed in the center of the playroom. Part III of the session comprised speeches (10 min). The experimenter asked the four children to sit in a semi-circle facing her. The experimenter then told the children that they were going to play a brief game of show-and-tell during which each child would stand up and tell the rest something about their recent birthday party. The experimenter then asked for a volunteer and allowed each child to stand and talk for up to 2 min. Following the speeches, the experimenter brought out to the center of the room a small table and four chairs and asked the children to sit around the table. A basket with colored cards was on the table and the experimenter asked the children to take one card of each color and make ve sets of cards (Part IV). Following this, the nal 15 min free-play began (Part V). The experimenter re-entered the room, brought back the box with the toys and allowed the children to play in the room by themselves for an additional 15 min. 1.4. Behavioral coding The videotapes of the play group sessions were coded using Rubins Play Observation Scale (POS; Rubin, 1989). Coders were blind to the hypotheses of the study. 1.4.1. Free-play sessions Ten-second intervals were coded for social participation (unoccupied, onlooking, solitary play, conversation, group play). This resulted in approximately 90 coding intervals per child in each of the two free-play sessions. Additional variables coded during the freeplay sessions included: (1) proportion of observational intervals that included the display of anxious behaviors (e.g. automanipulatives, digit

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sucking, crying); (2) the latency to the childs rst spontaneous utterance (free play session only); (3) the frequency of child initiated social interactions; and (4) the frequency of social interactions from peers. Of particular interest to this study was the measure of observed shyness during freeplay. This measure is computed by aggregating: proportion of unoccupied+onlooking behavior+proportion of anxious behaviors (Rubin, 1989). The inter-relations among these individual measures were examined before aggregating; all individual measures were highly related (P < 0.01). Each individual measure was then z-scored before aggregating. 1.4.2. Clean-up and ticket-sorting sessions During the clean-up and ticket sorting sessions, the proportion of time each child spent o taskunoccupied was recorded. Behaviors were considered o-task during the cleanup if they did not involve such actions as picking up the toys, or placing the toys in the toy box. O-task behaviors during the ticket sorting session included not sorting tickets or not talking about the task at hand. Unoccupied behavior was dened similarly to the behavioral variable of the same name used in the freeplay sessions. Time spent o-task but engaged in any other type of alternative activity (e.g. goong o, continuing to play with toys, disrupting others who were trying to cleanup/sort tickets), was coded as o-task-goong o. Of particular interest to the present study were individual measures of unoccupied behavior during clean-up and ticket sorting task and o-task-goong o. 1.4.3. Reliability The POS (Rubin, 1989) and additional variables were coded by four independent observers. Inter-rater reliability on a randomly selected group of children totaling 30% of the sample was calculated between pairs of observers using Cohens kappa. Kappas between pairs of raters ranged from K=0.60 to 0.80. Intercoder disagreements were resolved by review and discussion. 1.5. DNA preparation and genotyping Genomic DNA was prepared from buccal swabs (Epicentre Technologies, Madison, WI). Children were instructed by an investigator or parent to collect cheek cells by rolling a buccal brush rmly on the inside of the cheek, approximately 20 times on each side. The brushes were air dried for 1020 min then sent to the laboratory at NIH for extraction within 3 days. DNA was prepared by absorption to a bead matrix and heat elution according to the manufacturers instructions. 1.5.1. DRD4 The DRD4 gene exon III polymorphism was assayed by polymerase chain reaction (PCR) using the primers and reaction conditions described by Lichter, Barr, Kennedy, Van Tol, Kidd, and Livak (1993) combined with a hot start. The number of repeats was determined by electrophoresis through a 3.5% agarose gel and ethidium bromide staining. Allele frequencies were 6.2% for allele 2, 1.9% for allele 3, 68.3% for allele 4, 1.6% for allele 5, 0.3% for allele 6, 21.4% for allele 7, and 0.3% for allele 8. Based on previous results (Benjamin et al., 1996), genotypes were classied into two groups: short (S) for s/s and long (L) for s/l and l/l, where s indicates alleles with 25 repeats and l indicates alleles with 68 repeats. Indistinguishable results were

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obtained when genotypes were coded according to the presence or absence of the seven repeat allele (Ebstein et al., 1996) or as the sum of allele lengths. 1.5.2. 5-HTT The 5-HTT gene promoter polymorphism (5-HTTLPR) was analyzed by PCR amplication followed by agarose gel electrophoresis as described (Lesch et al., 1996). Allele frequencies were 59.0% for the short (s) allele and 41.0% for the long (l) allele; no extra-long alleles were observed in this population. Based on previous results (Lesch et al., 1996) genotypes were dichotomized as short (S) for s/s and s/l and long (L) for l/l. 1.5.3. 5-HT2C The 5-HT2C receptor gene codon 23 polymorphism was genotyped by PCR amplication, digestion with Hinf1 restriction endonuclease, and 3.0% agarose gel electrophoresis (Burnet et al., 1997; Lappalainen et al., 1995). Hinf1 digestion cleaves the Cys allele but not the Ser allele. Allele frequencies were 87.9% Cys and 12.1% Ser. 1.6. Data analysis We computed separate analyses of variance (ANOVAs) with DRD4 Group (short, long), 5HTT (short, long), and 5-HT2C (Cys, Ser) as between-subjects factor on each dependent measure. The dependent measures were: observed shyness and unoccupied behavior during cleanup and ticket sorting task and o-task-goong o behavior during peer play and the CBCL aggression and social adjustment subscales.

Fig. 1. Dierences between short and long repeat dopamine D4 receptor (DRD4) gene groups on maternal report of problems with childrens aggression at age four in normal development.

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2. Results 2.1. DRD4 gene and aggression We predicted that children with long versus short allele repeats of the DRD4 receptor gene would be rated by their mothers as having more problems with aggression and would exhibit more observed disruptive behaviors during peer play at age four. As predicted, the analysis revealed a signicant main eect for DRD4 group on the CBCL aggression subscale [F(1, 159)=4.25, P < 0.04]. Fig. 1 presents the between-subjects dierence on this measure. As can be seen in Fig. 1, children with long allele repeats of the DRD4 receptor gene were reported by their mothers to have signicantly more problems with aggression at age four compared with children with short allele repeats. The two DRD4 groupings were not distinguishable based on the observed aggression-related individual behavioral measures collected during peer play. Nor were the two DRD4 groupings distinguishable on the observed shyness measure or the CBCL social adjustment subscale. 2.2. Serotonin genes and shyness We predicted that children with short versus long genotypes of the 5-HTT transporter gene and the Ser23 versus Cys23 allele of the 5-HT2C receptor gene would be rated by their mothers as having more problems with social adjustment and would exhibit more observed shyness during peer play and at age four. We did not nd any signicant group dierences on the CBCL social adjustment measure or the observed shyness measure for the 5-HTT gene. As well, we did not nd any signicant group dierences on the CBCL aggression subscale or on the observed aggression-related individual behavioral measures for the 5-HTT gene. We also did not nd reliable dierences associated with the 5-HT2C gene, for which the Ser substitution had too low a frequency to give good sample sizes of individuals hemizygous or homozygous for the rare allele, precluding comparisons.

3. Discussion We found a signicant association of the DRD4 receptor gene with maternal report of problems with aggression in preschoolers. Children with long versus short allele repeats of the DRD4 receptor gene were rated by their mothers as having signicantly more problems with aggression at age four. This pattern of association is largely consistent with other studies which have noted an association between the DRD4 receptor gene and novelty seeking in human adults in which adults with long repeats of the DRD4 receptor gene scored higher on self-report measures of novelty seeking (Ebstein, Nemanov, et al., 1997; Noble et al., 1998; Ono et al., 1997; Strobel et al., 1999; Tomitaka et al., 1999). Novelty seeking is a precursor of aggression. What role does the DRD4 receptor gene play in childrens social behavior? It appears that the DRD4 gene may play some role in the dysregulation and disinhibition of cognitive and behavioral responses to stimulation. Dopamine has been implicated as a major neurotransmitter involved in euphoria and approach and reward seeking behaviors. Children who engage in aggressive and

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disruptive behaviors have problems with attending and staying on-task; such children as reported herein were more likely to have the longer versus shorter allele repeat of the DRD4 polymorphism. This nding is in line with other recent studies of children which have noted an association of the DRD4 receptor gene with attention decit and hyperactivity disorders (LaHoste et al., 1996; Swanson et al., 1998) and neonatal temperament (Ebstein et al., 1998). Children and newborns with the long versus short repeats are more likely to have problems with attention and hyperactivity and are likely to be highly reactive. Some of these motor features and o-task behaviors are similar to those displayed by the highly reactive infants (e.g. Kagan & Snidman, 1991) and exuberant children (Fox et al., 2001) described earlier. It is important to note that we did not nd any signicant associations of the DRD4 and the serotonin genes with the observed behavioral measures collected during peer play at age four, although we did nd a dierence on the maternal report measure. One possible explanation might be that mothers were more accurate in detecting temperamental and behavioral dierences in their children than our observational methods because mothers have the opportunity to observe their children throughout development for longer periods of time than our single observation at age four. It is also important to comment on the fact that we did not nd any signicant associations for the 5-HTT gene and our measures of shyness, while the adult literature has noted associations between this gene and anxiety-related problems. Given that shyness is a complex phenomenon that engenders a certain degree of cognitive maturation, it is likely that shyness and anxiety observed during the preschool years is not the same as in adults. Accordingly, the role of the 5-HTT in preschoolers shyness probably diers from that of adult neuroticism. The origins of human personality are undoubtedly complex. The present study appears to be the rst large scale investigation of children to consider maternal report as well as behavioral measures derived from direct observation in the study of the molecular genetics of personality development. The present ndings extend some previous work with adults to the preschool years and suggest that the DRD4 receptor gene may play some role in childrens aggression in normal development.

Acknowledgements This research was supported by a grant from the National Institutes of Health (HD 17899) awarded to Nathan A. Fox. The authors wish to thank the parents and children for their participation and Stacy Barton, Susan Calkins, Genevieve Erb, Lisa Perry-Moss, Ariana Shahinfar, and Cindy Smith for their help with behavioral data collection at Maryland.

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