+Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS The European Journal of Psychiatry xxx (xxxx) xxx---xxx www.elsevier.es/ejpsy ORIGINAL ARTICLE Monoamine oxidase A (MAOA) interaction with parenting practices on callous-unemotional traits in preschoolers N. Pueyo a , J.B. Navarro b , M. Fatjó-Vilas c , N. de la Osa d , E. Penelo b , L. Fañanás e , L. Ezpeleta d,∗ a Departament de Psicologia Clínica i de la Salut, Universitat Autònoma de Barcelona, Spain b Departament de Psicobiologia i de Metodologia de les Ciències de la Salut, Unitat d’Epidemiologia i de Diagnòstic en Psicopatologia del Desenvolupament, Universitat Autònoma de Barcelona, Spain c Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals Facultat de Biologia, Institut de Biomedicina de la Universitat de Barcelona (IBUB), FIDMAG Germanes Hospitalàries Research Foundation, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Universitat de Barcelona, Spain d Departament de Psicologia Clínica i de la Salut, Unitat d’Epidemiologia i de Diagnòstic en Psicopatologia del Desenvolupament, Universitat Autònoma de Barcelona, Spain e Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Facultat de Biologia, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Universitat de Barcelona, Spain Received 7 November 2020; accepted 1 February 2021 KEYWORDS Abstract Callous unemotional Background and Objectives: From a gene-by-environment perspective, parenting in interaction traits; with the polymorphism in the Monoamine oxidase A (MAOA) gene (MAOA-uVNTR) might also Development; be associated with increased callous-unemotional traits (CU) in preschoolers. MAOA-uVNTR Gene by results in differential enzyme activity, so that high-activity alleles (MAOA-H) are linked to environment; reduced dopamine, serotonin, and norepinephrine availability in comparison to low-activity MAOA; allele (MAOA-L). As MAOA-uVNTR has been previously described to moderate the relationship Parenting practices; between childhood parental maltreatment and aggressive and antisocial behavior, it may also Preschool play a role in CU traits etiology. ∗ Corresponding author at: Departament de Psicologia Clínica i de la Salut. Edifici B. Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. E-mail addresses: natalia.pueyo@e-campus.uab.cat (N. Pueyo), JoseBlas.Navarro@uab.cat (J.B. Navarro), mfatjo-vilas@fidmag.com (M. Fatjó-Vilas), Nuria.delaosa@uab.cat (N. de la Osa), eva.penelo@uab.cat (E. Penelo), lfananas@ub.edu (L. Fañanás), Lourdes.Ezpeleta@uab.cat (L. Ezpeleta). https://doi.org/10.1016/j.ejpsy.2021.02.003 0213-6163/© 2021 Asociación Universitaria de Zaragoza para el Progreso de la Psiquiatrı́a y la Salud Mental. Published by Elsevier España, S.L.U. All rights reserved. Please cite this article as: N. Pueyo, J.B. Navarro, M. Fatjó-Vilas et al., Monoamine oxidase A (MAOA) interac- tion with parenting practices on callous-unemotional traits in preschoolers, The European Journal of Psychiatry, https://doi.org/10.1016/j.ejpsy.2021.02.003 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS N. Pueyo, J.B. Navarro, M. Fatjó-Vilas et al. Methods: Data was collected through questionnaires answered by parents and teachers. MAOA- uVNTR was genotyped in 368 Caucasian children from a community sample (51.9% male). Multiple linear regression analyses were conducted to analyze the interaction effect of MAOA genotypes and both positive parenting and punitive parenting practices on CU traits at two different periods (3 and 5 years old) and separately by sex. Results: No significant interactions were found for boys. Among girls, a significant interaction effect was found for MAOA-LL carriers, who showed higher CU traits at age 5 when exposed to higher punitive or positive parenting at age 3. Conclusions: Our study provides the first evidence for significant MAOA × early parenting effects on CU traits in preschoolers, specifically among female MAOA-LL carriers. This suggests that the MAOA-LL genotype for girls is associated with higher sensitivity to both positive and punitive parenting in girls, so that MAOA-LL emerges as a genotype that confers higher vulnerability to parental influences. © 2021 Asociación Universitaria de Zaragoza para el Progreso de la Psiquiatrı́a y la Salud Mental. Published by Elsevier España, S.L.U. All rights reserved. Introduction an individual’s sensitivity towards social environments, such as parenting. While one study found a salient G × E interac- Callous Unemotional (CU) traits are seen as precursors of tion in CU trait development on BDNF and harsh parenting,9 adult psychopathy and have been added to the DSM-5 as a the question arises whether other candidate genes for CU specifier to diagnose conduct disorder under the term ‘lim- traits might also moderate the effect of parenting practices ited prosocial emotions’ (LPE) in order to identify a subgroup on CU traits development. of children and adolescents who show a distinct prosocial To provide more insight into the complex relationship and emotional functioning such as lack of empathy, lack of between genes, parenting practices and CU trait develop- guilt and deficits in emotional expression.1 ment, the current study focuses on Monoamine Oxidase A CU traits are highly heritable, and a meta-analysis esti- gene (MAOA). Moore et al.10 identified, among other candi- mated that genetic factors account for 42% and 68% of the date genes for CU traits, MAOA. This gene encodes for the variation of CU traits.2 Takahashi et al.3 found that the Monoamine Oxidase A enzyme (MAO-A) that catalyzes the genetic effect on CU traits varies depending on the develop- degradation of brain neurotransmitters such as serotonin, mental path of CU traits, so that childhood-onset CU traits dopamine, and norepinephrine.11 Deficits in the serotonin (around age 7) seem to be under a higher genetic influence system have been associated with CU traits,12 but the spe- than CU traits that develop later across adolescence. More- cific role of MAOA in the etiology of CU traits has remained over, they indicate that the course of CU traits seems to unexamined. be dynamic, with environmental influences accounting for The MAOA gene has a variable number of tandem repeats 23.5% of the variance of initial CU traits, but for 56.4% in (uVNTR) polymorphism in its promoter sequence. The dif- the stability of these traits. Among the environmental fac- ferent allelic variants of this polymorphism are associated tors that influence the development of childhood CU traits, with changes in the transcriptional efficiency of the gene, parenting practices have been the focus of most studies.4 which results in low and high enzymatic activity alleles Harsh, inconsistent parenting and corporal punishment have (MAOA-L and MAOA-H, respectively).13 As the MAOA gene also been identified as risk factors for increases in CU traits is located on the X chromosome (Xp11.23), males inherit in pre-schoolers.5 At the same time, positive parenting can a single allele and are therefore hemizygous for either be considered a protective factor and strategies such as pos- MAOA-L or MAOA-H, whereas females can be homozy- itive reinforcement, parental sensitivity and warmth have gous (MAOA-LL/MAOA-HH) or heterozygous (MAOA-HL). The shown to predict lower CU levels among children.6 lower MAO-A activity results in different neurochemical, Certainly, not all children are equally vulnerable to neural and behavioral alterations14 and has been identified certain parenting practices in the development of CU as the ‘‘risk allele’’ for antisocial behavior15 and aggressive traits. Some research suggests that sensitivity to parent- behavior,16 especially among males. MAOA-L has also been ing practices might be explained through individual genetic associated with CU traits in male adolescents with comorbid variability.7 In this sense, Gene by Environment (G × E) inter- attention deficit/hyperactivity disorder.17 Thus, neuroimag- action studies have focused on the diathesis-stress model ing studies have shown that the MAOA-L allele has an impact and have found that certain genotypes confer vulnerabil- on altering neural circuits such as the amygdala or the pre- ity to adverse environments. From another perspective, the frontal cortex, which are implicated in aggressive behavior differential susceptibility model8 suggests individuals might and emotional processing.18 be more susceptible to adverse parenting styles, but at the Research also indicates the existence of a robust sex- same time, might also benefit more from positive parenting dependent G × E interaction on MAOA-L and childhood practices. Both models show that genetic influences shape maltreatment, showing that males who carry the MAOA- 2 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS The European Journal of Psychiatry xxx (xxxx) xxx---xxx L allele and are exposed to abuse or maltreatment also Table 1 Characteristics of the Sample (N = 368). develop more antisocial behavior19 and conduct disorder.20 Studies on females are less frequent and present less robust Sex; n (%) Male 191 (51.9) findings, but suggest that MAOA-HH confers vulnerability Socioeconomic status; n (%) High 134 (36.4) towards adversity, resulting in the ‘‘risk allele’’ for anti- Middle 163 (44.3) social behavior.15 Low 71 (19.3) Based on previous G × E studies, we hypothesized the One-parent family; n (%) 17 (4.6) presence of sex dependent G × E interactions on CU trait Age of the parents; mean (SD) Mother 36.7 (4.1) development on preschoolers, so that boys who carried the Father 39.2 (5.4) MAOA-L allele and girls who carried the MAOA-HH allele and who experienced punitive parenting styles would exhibit higher levels of CU traits. At the same time, these chil- were 3 and 5 years old. The ICU is a 24-item and 4-point dren would show lower levels of CU traits when exposed to Likert scale questionnaire that assesses CU traits, and the positive parenting practices. Moreover, we examined these total score was used. In our sample, Cronbach’s alpha for G × E interactions on CU traits at two different periods the total score at both 3 years old and 5 years old was .90. (ages 3 and 5), which represent initial and ending points Genotype. Genomic DNA was extracted from children’s of preschool age. Preschool age is a developmental period buccal mucosa on a cotton swab using the Real Extraction in which empathy, emotional expression and conscience DNA Kit (Durviz S.L.U., Valencia, Spain). The Polymerase emerge, making it an important time in the pathway to Chain Reaction (PCR) was carried out using 1 ␮l of DNA and early CU traits.21 During these years, children are very sen- 14 ␮l of mix. The cycling parameters of the PCR were as fol- sitive to parenting practices and research has shown that lows: an initialization step at 94 ◦ C for 2 min, followed by positive parenting practices protect children from socioemo- 30 cycles of denaturation at 94 ◦ C, annealing at 66 ◦ C for tional difficulties, while harsh practices increase the risk of 1 min, extension at 72 ◦ C for 1 min and a final elongation developing externalizing problems and CU traits.22 Because at 72 ◦ C for 15 min. The primers used were MAOA-Forward: genetic influences seem to be more important at earlier ages 5 -ACA GCC TGA CCG TGG AGA AG-3 (marked with fluo- than later in development,23 we hypothesized that G × E rochrome HEX) and MAOA-Reverse: 5 -GAA CGG ACG CTC interactions would be different across age.3 CAT TCG GA-3 . 1 ␮l of the resulting amplified DNA was mixed with 10 ␮l of HI-DI formamide and 0.4 ␮l of ROX and kept at 95 ◦ C for 5 min before being put in the freezer for 1 min. The Material and methods uVNTR polymorphism of the MAOA gene was genotyped using GeneMapper® Software v4.1. The genotyping success rate Participants was 92.5% (N = 368), leaving 33 individuals with an undeter- mined uVNTR polymorphism. Ten per cent of the individuals In the context of a longitudinal study of psychological risk were randomly selected for re-genotyping to confirm the factors during development, a random sample of 2,283 validity and accuracy of the method. This re-testing showed children from the census of preschoolers in grade P3 (3- 100% reproducibility. Regarding the Hardy-Weinberg equi- year-olds) in Barcelona (Catalonia, Spain) were screened librium, the genotype of the MAOA activity for women in for behavioral problems.24 This began with an initial screen- the sample (n = 177) was in equilibrium (␹2 = 0.097, p = .95). ing using the parent-administered Strengths and Difficulties There was no need to test the equilibrium for males since Questionnaire (SDQ)25 enriched with four additional oppo- their genotype distribution is the same as their allelic dis- sitional defiant disorder items to complete the DSM-IV tribution (they only have one copy of the MAOA gene). description. A total of 1,341 families (58.7%) agreed to par- In line with previous studies, the MAOA genotypes ticipate. In a second stage of the sampling, all the children were grouped according to their functionality.19,27 The who screened positively for behavioral problems and an low-activity MAOA genotype includes individuals with the additional 30% of the children with negative screening scores 3-repeat allele, whereas the high-activity MAOA genotype continued and were assessed annually. Of those included, includes participants with 3.5, 4 or 5 repeats. 622 families (89.4 %) agreed to participate further. No statis- tically significant differences in sex (p = .82) or type of school Environmental variables (p = .85) between participants and drop-outs were found. For The environmental variables were measured when the chil- the present study, which corresponds to a prospective design dren were 3 years old. with independent variables assessed at age 3 and dependent Parenting Practices. The Alabama Parenting Question- variables assessed at ages 3 and 5, only Caucasian children naire Preschool Revision (APQ-Pr)28,29 consists of 24-items were included, to control possible ethnic or racial variations on a 5-point Likert scale which measures three dimensions in MAOA allele frequencies.13 MAOA genotype was available of parenting: positive parenting, inconsistent parenting and for 368 children (59.2%). Table 1 presents the demographic punitive parenting. Positive parenting (12 items) and puni- information at age 3. tive parenting (5 items) scores were answered by parents when the children were 3 years old (5.8% father, 48.5% Materials mother, 44.7% both), were taken into consideration. The positive parenting subscale measures how frequently the Individual variables parent interacts in games and shared time and how often CU traits outcome. The Inventory of Callous-Unemotional they use positive reinforcement to foster appropriate behav- Traits (ICU)26 was answered by teachers when the children ior. The punitive parenting subscale measures how often the 3 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS N. Pueyo, J.B. Navarro, M. Fatjó-Vilas et al. Table 2 Zero-order correlations between ICU scores at ages 3 and 5, parental styles, SES and SDQ-conduct problems at age 3, separately by sex. 1. 2. 3. 4. 5. 6. 1. ICU at age 3 .35* .12 .18* .07 .54* 2. ICU at age 5 .34* .07 .03 .08 .13 3. APQ-Pr Punitive parenting .09 .04 −.14 −.05 .13 4. APQ-Pr Positive parenting −.15* .06 −.21* −.13 .13 5. Socioeconomic status .04 −.03 .08 .04 .01 6. SDQ-conduct problems .57* .13 .21* −.17* .03 Above diagonal correlations for boys. Below diagonal correlations for girls. *p < .05. parent spanks, slaps or yells at their children to punish inap- the mean of the two quantitative environmental variables. propriate behavior.30 The internal consistency in our sample The SDQ conduct problems scale and socioeconomic sta- showed an acceptable value for positive parenting (Cron- tus (SES) were included in all models as adjusting terms at bach’s alpha = .75) but a low value for punitive parenting baseline (age 3). The two measures of parenting style were (alpha = .42). As both scales had few items (6 for positive retained in the model although its interactions had been parenting and 3 for corporal punishment) and most of them deleted. Additionally, the ICU score at age 3 was included showed skewed distributions, inter-item mean correlation as a covariable in models predicting ICU score at age 5. was also calculated, resulting in acceptable values of r = .31 Normality of the dependent variable (ICU total score) was for positive parenting and r = .25 for punitive parenting. verified separately for boys and girls at age 3 and 5 using The Strengths and Difficulties Questionnaire (SDQ)25 is a two graphical inspection techniques, boxplot and standard- 25-item screening questionnaire for child behavior and emo- ized normal probability plot. Inspection of the boxplot also tional problems. Teachers answered the questionnaire when confirmed normality of residuals for each regression model the children were 3 years old and the conduct problems sub- estimated. scale (Ordinal alpha = .85) was introduced as an adjusting term in linear regression models. Results Procedure Table 2 shows the zero-order Pearson correlations between CU traits at ages 3 and 5, parenting, SES and conduct prob- The study was approved by the ethics review committee lems, separately for boys and girls. The highest positive of the author’s institution. Schools were informed and the associations were between CU traits and conduct problems participating parents had to provide written consent. The at age 3, and between the two CU traits measures. There families who met the inclusion criteria and were willing to were some relevant differences among sexes. The relation- participate were contacted by telephone and interviewed at ship between CU traits at age 3 and positive parenting was the school. The questionnaires were administered at the end direct for boys, but inverse for girls. Also, the associa- of the course to guarantee that teachers knew the children tion between punitive parenting and conduct problems was they were evaluating well. stronger for girls than for boys. Statistical analysis The data was analyzed using STATA 16.0 for Windows. The Allelic and genotypic frequencies and distribution Type I error was fixed at .05. To compare means of APQ-Pr of environmental factors between genotypes, Student’s t-tests were calculated for boys, while analysis of variance with post-hoc comparisons The H allele was present in 69.1% (n = 132) of the boys and and Bonferroni correction for multiple comparisons was esti- the L in 30.9% (n = 59). The HH genotype was present in mated for girls. 48.6% (n = 86) of the girls, HL in 42.9% (n = 76) and LL in 8.5% The G × E analyses were conducted using separate mul- (n = 15). Table 3 shows that there were no statistically signifi- tiple linear regressions for each sex with the dependent cant differences in the two environmental scores considered variable being ICU scores at 3 and 5 years old (4 regres- in relation to the genotypes for either boys or girls. sion models in total). The terms entered in each model as independent variables were MAOA alleles, APQ-Pr Positive and APQ-Pr Punitive measured at age 3, and the first-order G × E interactions on CU traits interactions terms between MAOA genotypes and the two environmental characteristics. Non-significant interactions Table 4 shows the results of the linear regressions modelling were removed from the model and in that case main effects CU traits at ages 3 and 5 from MAOA, APQ-Pr Positive and coefficients were reported. Conversely, in the presence of APQ-Pr Punitive (measured when the children were 3 years significant interaction, simple effects of each environmen- old) and its interaction separately for boys and girls. No sta- tal variable were calculated separately for each genotype, tistically significant effect was found on CU traits at age while differences between genotypes were calculated for 3. 4 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS The European Journal of Psychiatry xxx (xxxx) xxx---xxx Table 3 Distribution of environmental factors at age 3 by sex and genotype. Boys (n=191) Girls (n = 177) H L (n = 59) p (H vs L) HH HL LL p (HH vs p (HH vs p (HL vs (n = 132) (n = 86) (n = 76) (n = 15) HL) LL) LL) APQ-Pr Parenting practices Punitive; mean 3.73 3.63 .740 3.89 3.46 3.07 .384 .305 1 (SD) (1.94) (1.87) (1.96) (1.64) (1.53) Positive; mean 41.06 40.46 .338 41.69 40.17 39.87 .054 .323 1 (SD) (3.91) (4.11) (3.80) (3.80) (6.08) Table 4 MAOAx Parenting (at age 3) results on ICU scores for boys and girls at ages 3 and 5. Boys Girls Response: ICU at age 3 B p 95% CI (B) B p 95% CI (B) APQ-Pr Punitive × MAOA .816 .576 APQ-Pr Positive × MAOA .250 .092 APQ-Pr Punitive parenting 0.35 .349 −0.39; 1.10 0.02 .948 −0.67; 0.72 APQ-Pr Positive parenting 0.26 .123 −0.07; 0.60 −0.07 .576 −0.33; 0.19 MAOA: L/LL vs. H/HH −1.31 .373 −4.21; 1.59 3.16 .059 −0.12; 6.45 HL vs. HH – – – −0.01 .996 −2.61; 2.60 Response: ICU at age 5 B p 95% CI (B) B p 95% CI (B) APQ-Pr Punitive × MAOA .906 <.001 APQ-Pr Positive × MAOA .126 .002 APQ-Pr Punitive parenting 0.14 .697 −0.56; 0.84 for HH 0.65 .283 −0.54; 1.84 for HL −0.49 .453 −1.77; 0.79 for LL 4.17 <.001 2.26; 6.08 APQ-Pr Positive parenting −0.03 .872 −0.44; 0.37 for HH 0.02 .954 −0.55; 0.58 for HL 0.06 .846 −0.51; 0.63 for LL 1.20 <.001 0.71; 1.68 MAOA: L/LL vs. H/HH −0.07 .969 −3.35; 3.21 8.05 <.001 3.82; 12.28 HL vs. HH – – – 0.67 .652 −2.27; 3.61 Boys are hemizygous H or L; All regression coefficients are adjusted by SDQ conduct problems scale and SES, additionally models at age 5 are adjusted by ICU score at age 3; the B column shows main effect in absence of significant interaction, and simple effects when interaction is significant. Bold values signifies the values are significant. The prediction of CU traits at age 5 shows non-significant Discussion parenting effects (measured at age 3) for boys, but signif- icant differences for girls. In the analysis of CU for girls at We analyzed the G × E interaction hypothesis that MAOA age 5, there was evidence of an interaction between puni- polymorphism moderates the impact of parenting practices tive parenting (APQ-Pr Punitive) and MAOA gene (p < .001). at age 3 on the risk of CU traits in male and female preschool- Higher levels of punitive parenting at age 3 were associ- ers at two different stages (ages 3 and 5). Contrary to our ated with higher levels of CU traits only in the LL genotype hypothesis, we found that the effect of early parenting prac- subgroup (p < .001) (Fig. 1A). An interaction with positive tices on CU traits is not moderated by MAOA alleles for parenting (APQ-Pr Positive) at age 3 was also detected boys, but it is for girls at age 5. In line with our hypothesis when predicting CU at age 5 in the group of girls (p = .002) on age specific G × E interaction in later stages, we found (Fig. 1B). Increased scores in APQ-Pr Positive parenting (age a salient G × E interaction only among girls at age 5. This 3) lead to significantly higher CU scores at age 5 only for is in line with previous research that found that age and the LL genotype (p < .001). The effect of increasing punitive sex function as moderating factors of CU traits on parenting parenting at age 3 on CU traits at age 5 for LL genotype practices.6 In our study, we moreover include genetic vul- (B = 4.17) was larger than the effect of increasing positive nerability in the etiology model, suggesting that girls who parenting (B = 1.20). carry the MAOA-LL genotype show higher CU traits at age 5 5 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS N. Pueyo, J.B. Navarro, M. Fatjó-Vilas et al. on vulnerability towards certain environments might be moderated by gender.34 Thus, we studied MAOA, which is an X-linked gene and operates differently in males and females.35 Females can be heterozygous and might undergo an X-inactivation of one of the alleles and show allelic expression of only one of the two alleles.36 Therefore, understanding the effects of MAOA is complicated as it is unclear if one allele is inactivated or not, which leads to sex differences in MAOA product.36 Moreover, G × E interactions might be under the differential effect on gender through the impact of different hormones in males and females, such as testosterone.32 Finally, the MAOA promoter region also revealed to be affected by an epigenetic mechanism that involves a chemical modification to the DNA which is called DNA methylation.37 This mechanism can modify gene expression and is considered a risk for mental disorders.38 As studies on specific MAOA promoter methylation have identi- fied higher methylation in females, especially among those with the low activity genotype,39,40 we cannot exclude the possibility that DNA methylation influenced our findings. Although findings are inconsistent, most studies sup- port MAOA-HH as the risk allele for females on antisocial behavior.15 Nonetheless, we report a MAOA-LL genotype- specific role on the increase in CU traits in 5-years old girls in interaction with early parenting practices. Our results would be in line with the alternative stream of studies that have Figure 1 Regression lines for effect of APQ-Pr Punitive (A) and identified MAOA-LL as the risk allele for antisocial behav- APQ-Pr Positive (B) at age 3 on ICU-5 years for girls depending ior among females.41,42 As this is the first study to include on MAOA. females in a MAOA x Environment interaction study on CU A. APQ-Pr Punitive. traits, our findings should be interpreted with caution and B. APQ-Pr Positive. should be replicated to clarify which alleles might confer vulnerability towards environment in the development of when exposed to punitive and positive parenting at age 3. CU traits among girls. This suggests that the MAOA-LL genotype for girls is asso- In our study, the MAOA-LL x Punitive parenting interac- ciated with higher sensitivity to both positive and punitive tion on girls increased CU traits at age 5, which would be parenting in girls, so that MAOA-LL and not the hypothesized consistent with previous research that associates harsh par- MAOA-HH allele, emerges as a genotype that confers higher enting with CU traits.2 When parents engage in physical and vulnerability to environmental influences. Interestingly, the verbal abuse, communicate poorly and distance themselves effect of punitive parenting on CU trait development among from their children, this directly influences the child’s ability female MAOA-LL carriers was three times higher than the to understand and interpret emotions and social situations.22 effect of positive parenting. Thus, when children experience harsh parenting or low All in all, we might fail to replicate the expected G × E parental warmth, they might react with negativity or aggres- interactions because the studies on which we have built our sion towards their parents. In turn, children with CU traits hypothesis were conducted mostly in children who experi- also seem to elicit more punitive parenting practices from enced severe early childhood experiences.20 As the cited their parents, resulting in a bidirectional influence between MAOA x Early adversity interactions might be of a spe- harsh parenting practices and child CU traits.43 At the same cific nature, it is possible that MAOA acts as a moderating time, not all children are equally sensitive towards parent- factor only when children experience extreme forms of ing practices, so that identifying a subgroup of children (in maltreatment or trauma,31 but not when they face less puni- our case, girls at age 5 who carry MAOA-LL alleles) can tive environments such as parenting practices. Moreover, help explain the biological vulnerability towards environ- most MAOA x Early adversity interaction studies focused mental factors and its effect on CU traits.10 Thus, our study on antisocial behavior as an outcome, and only Fowler has showed that girls who experience punitive parenting et al.17 specifically addressed CU traits in their MAOA x Early and carry the MAOA-LL allele increment almost three times adversity interaction study. Therefore, our novel and coun- more their CU traits than those who are exposed to positive terintuitive findings could be explained by the fact that parenting. This suggests that early harsh parenting behav- antisocial behavior and CU traits are different constructs iors have a deeper impact on girl’s CU traits than positive that may have distinct underlying G × E interactions.32 parentings. In line with developmental child psychopathol- The sex specific G × E interaction in our study could be ogy theories, early harsh parenting has severe long-term explained by sex differences in heritability of CU traits. effects on the biological and psychophysiological reactivity Boys seem to be under greater genetic influences than girls, of children and impacts their stress response systems.44 Chil- whereas for girls the influence of environmental factors dren that have experienced punitive or coercive parenting is higher.33 At the same time, the individual differences practices might show higher levels of arousal and anxiety, 6 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS The European Journal of Psychiatry xxx (xxxx) xxx---xxx as well as altered reward-processing and fear-processing a control covariate in our regression models. CU traits are systems.44,45 As such, punitive parenting also predicts CU often comorbid with antisocial behavior or CD, so further traits from early childhood on, but positive parenting, in isolation of the possible effect of CD provides more accurate contrast, seems to have a more deteriorating effect on MAOA x E interaction models of CU traits. Also, the socioeco- CU traits.22 Interestingly, the MAOA-LL x Positive parenting nomic status of the families was used as a control covariate. interaction predicts higher CU traits among girls at age 5. Finally, this study included positive and negative environ- Even though positive parenting is generally considered to mental factors in the G × E interaction models because most prevent and reduce the risk of CU trait development among of the MAOA x E interaction studies on CU traits to date preschoolers and children,45 studies have also shown that have studied genetic effects on exposure to strongly aversive among preschoolers, positive parenting strategies can pre- environmental factors such as negative life events or harsh dict CU traits.6 It might be that parents of children with parenting styles.17,48 Hence, studying positive and less aver- CU traits engage in more positive parenting practices such sive contexts helps to clarify how these G × E interactions as parental warmth or giving rewards to respond to their work in both environments. challenging children’s CU behavior. Nevertheless, the present study also has some notable Moreover, girls might be under a greater parental influ- limitations that should be considered. First, while our results ence, as they are generally more closely monitored by their are in line with previous studies focused on the MAOA parents.46 Daughters are also treated with more reasoning gene,20,49 we are aware of the methodological and statis- and dialogue, whereas sons experience more authoritar- tical concerns that gene by environment (G × E) interaction ian parenting practices47 and parents might be more prone studies have raised, such as small effect sizes and limited to respond at daughter’s behavior with positive parenting statistical power.50 In this regard, although our total sample strategies. Thus, early CU trait behavior of girls might elicit size (n = 368) is larger than other studies on G × E interaction both punitive and positive parenting practices at age 3, on CU traits,9 it might not have enough power to estimate which, at the same time, are moderated by MAOA-LL on small effects. Second, we cannot rule out the possibility that CU traits at age 5. G-E correlations (rGE) might explain our findings,20 so that Also, parents that have to take care of children with CU our G × E interaction on girls might be mediated by passive traits show higher parental inconsistencies and change their rGE (parents transmit to their daughters a genetic suscepti- parental strategies over time.6 Applied to our study, this bility towards CU traits) or evocative rGE (girls with a certain would suggest that parents might initially have started with genotype may show CU traits that traits elicit punitive par- more positive reinforcement and parental warmth at age 3 enting). Future research should test for the presence of rGE to counter their daughter’s emerging CU behavior,43 but, at in the MAOAx Parenting interactions on CU traits. Third, our the same time, they also adopted more punitive practices. G × E design has focused only on one specific environmental This would explain why girls at age 5 carrying MAOA-LL alle- factor (parenting), while there are other environmental fac- les showed higher CU traits when exposed to both positive tors that might be influencing CU traits.4 Therefore, Gard and negative parenting at age 3. In that sense, our find- et al. suggest51 that further studies should address more ings could then be an indicator of the dynamic nature of complex relationships between multiple environment (G × parenting practices and its reciprocal effect on CU traits, E × E) or multiple genes (G × G × E). Thus, the authors moderated by genetic vulnerabilities. highlight novel and more sophisticated molecular genetic Finally, the lack of interaction at age 3 across sex might approaches such as neurogenetics which provide promis- be explained by the fact that early childhood onset CU traits ing results to find polygenic risk scores, instead of focusing are under a higher genetic influence than later developed on a single candidate gene as the present study does. Also CU traits.3 This builds on current research that analyzes Imaging G × E interaction studies are of interest in gaining a genetic effect changes over time and according to differ- deeper understanding of how neural alterations mediate the ent CU developing paths, in which early emerging CU traits effects of G × E interactions to psychopathology.51 Fourth, show higher heritability. This is in line with our findings, there is a small number of girls in the LL genotype due to because CU traits at age 3 (early onset CU traits) showed the usual genotypic distribution. Fifth, parenting practices no G × E interaction, while CU traits at age 5 among girls were measured using self-reports which may be under the (later-on CU traits) can be predicted by a G × E interaction. effect of distortions such as social desirability or individual In our sample, it seems that it is later in development (at interpretation of the items. Sixth, the study focuses on a age 5) when the interplay of gender, genetic vulnerability short period of time (ages 3 and 5), so it is unclear which and environment on CU traits becomes salient. G × E interactions could be present in later childhood devel- This study has several key strengths. It includes a opment, as the effect of environmental factors may vary prospective G × E design, with repeated assessment of CU according to the timing of the experiences.32 traits. As early childhood is a period when children are very All in all, the results indicate that the influence of the sensitive to emotion regulation, our study permitted test- MAOA x Parenting on CU traits is sex- and age-specific. If ing whether early life environments (parenting practices) at replicated, our study suggests that early parenting expe- different times were associated with the development of riences at age 3 might have long-term effects, resulting CU traits. Moreover, while most of the G × E research on CU in a sex-specific G × E interaction for girls in later phases has focused on high-risk children who have experienced mal- of development. Understanding how genes might interact treatment, our study used data from a community sample with parenting practices in early childhood is crucial in of preschoolers to study how MAOA interacts in less aversive preventing early CU symptoms from developing into more environments on CU trait development. Another strength of severe forms of conduct disorder or antisocial behavior,45 as this study is the use of the SDQ conduct problems scale as parenting strategies are among the most salient risk fac- 7 +Model EJPSY-138; No. of Pages 9 ARTICLE IN PRESS N. Pueyo, J.B. Navarro, M. Fatjó-Vilas et al. tors. Thus, treatment models of CU traits often focuses 5. Flom M, White D, Ganiban J, Saudino KJ. Longitudinal on cognitive-behavior strategies and parent-child interven- links between callous-unemotional behaviors and parent- tions, with treatment outcomes that are generally poor or ing in early childhood: a genetically informed design. limited.2 Underlying G × E mechanisms might contribute to J Am Acad Child Adolesc Psychiatry. 2020;59(3):401---9, explain why some children show a worse treating response http://dx.doi.org/10.1016/j.jaac.2019.03.013. 6. Hawes DJ, Dadds MR, Frost ADJ, Hasking PA. Do child- than others, so that children carrying certain risk alle- hood callous-unemotional traits drive change in parenting les might be more sensitive than others towards parenting practices? J Clin Child Adolesc Psychol. 2011;40(4):507---18, strategies.7 These G × E mechanisms of CU traits develop- http://dx.doi.org/10.1080/15374416.2011.581624. ment might need to be considered in the process of designing 7. Pluess M, Belsky J. Differential susceptibility to maternal sen- effective treatment interventions. For example, interven- sitivity. In: In: Maternal Sensitivity: A Scientific Foundation for tions on parent-child interactions in clinical settings should Practice. University Louisville: Press, Nova; 2011. p. 95---106. take into consideration possible sex-differences and specif- 8. Belsky J, Jonassaint C, Pluess M, Stanton M, Brummett ically address parent-girl relationships when CU behaviors B, Williams R. Vulnerability genes or plasticity genes? appear already in early childhood. Mol Psychiatry. 2009;14(8):746---54, http://dx.doi.org/ As G × E interactions might have cascading influences on 10.1038/mp.2009.44. 9. Willoughby MT, Mills-Koonce WR, Propper CB, Waschbusch development and stability of CU traits in older children,3 DA. Observed parenting behaviors interact with a poly- longitudinal approaches need further exploration in order to morphism of the brain-derived neurotrophic factor gene analyze the underlying mechanisms that could shape differ- to predict the emergence of oppositional defiant and ent developing pathways to CU traits. Thus, our approach callous-unemotional behaviors at age 3 years. Dev Psy- might help to identify a group of children who are more chopathol. 2013;25 4 Pt 1:903---17, http://dx.doi.org/10.1017/ vulnerable to their environment in a certain developmen- S0954579413000266. tal period, providing insight on individual differences in the 10. Moore AA, Blair RJ, Hettema JM, Roberson-Nay R. The development of CU traits. genetic underpinnings of callous-unemotional traits: a system- Ethical considerations atic research review. Neurosci Biobehav Rev. 2019;100:85---97, http://dx.doi.org/10.1016/j.neubiorev.2019.02.018. The authors assert that all procedures contributing to 11. Weyler W, YP H, XO B. Biochemistry and genetics of this work comply with the ethical standards of the relevant monoamine oxidase. Pharmacol Ther. 1990;47:391---417, national and institutional committees on human experimen- http://dx.doi.org/10.1016/0163-7258(90)90064-9. tation and with the Helsinki Declaration of 1975, as revised 12. Moul C, Dobson-Stone C, Brennan J, Hawes DJ, Dadds in 2008. MR. Serotonin 1B receptor gene (HTR1B) methylation as a risk factor for callous-unemotional traits in anti- Funding social boys. PLoS One. 2015;10(5):1---15, http://dx.doi. org/10.1371/journal.pone.0126903. 13. Sabol S, Hu S, Hamer D. A functional polymorphism in This work was supported by the Spanish Ministry of Science, the monoamine oxidase A gene promoter. Hum Genet. Innovation and Universities [Grant PGC2018-095239-B-I00 1998;103:273---9, http://dx.doi.org/10.1007/s004390050816. (MICIU/FEDER)]. We also acknowledge the support of 14. Godar SC, Fite PJ, Mcfarlin KM, Bortolato M. 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