Childhood adversity and psychosis: Examining whether the association is due to genetic confounding using a monozygotic twin differences approach

Abstract Purpose To test whether the association between childhood adversity and positive and negative psychotic experiences is due to genetic confounding. Method Childhood adversity and psychotic experiences were assessed in an ongoing sample of 226 twins from the general population. A monozygotic (MZ) twin differences approach was used to assess possible genetic confounding. Results In the whole sample, childhood adversity was significantly associated with positive (β = 45; SE = 0.16; P = 0.008) and negative psychotic experiences (β = 0.77; SE = 0.18; P < 0.01). Within-pair MZ twin differences in exposure to childhood adversity were significantly associated with differences in positive (β = 71; SE = 0.29; P = 0.016) and negative psychotic experiences (β = 98; SE = 0.38; P = 0.014) in a subsample of 85 MZ twin pairs. Conclusions Individuals exposed to childhood adversity are more likely to report psychotic experiences. Furthermore, our findings indicate that this association is not due to genetic confounding.


Introduction
A growing body of research indicates that attenuated psychotic experiences are present in a substantial proportion of healthy individuals [26,44,6]. This evidence supports the conceptualization of psychosis as a continous trait, the distribution of which extends into the general population [25,42]. In the absence of illness and need of treatment, these milder forms of psychotic symptoms are referred to as psychotic experiences [25]. The study of the risk factors underlying the expression of psychotic experiences can greatly contribute to the understanding of psychotic disorders because it has been shown that: psychotic experiences precede the onset of psychosis, thus psychotic experiences can help to identify subjects at risk [26,14] and; clinical and subclinical psychotic symptoms are likely to involve common risk factors in their etiology [26,44,25]. In this context, childhood adversity constitutes an environmental risk factor which has been frequently related to the expression of both clinical [7,8] and subclinical psychotic symptoms or psychotic experiences [27,38].
Interestingly, despite the efforts made in the genetics of psychotic disorders in the last decades, a growing body of research points toward a contribution of environmental factors, including childhood adversity, to their etiology [45,43,49]. Furthermore, Van Os et al. [45] have recently pointed out that genetic factors involved in these disorders are likely to operate via environmental factors by making individuals more sensitive (gene-environment interaction) or prone (gene-environment correlation) to certain environments [41]. These mechanisms of gene-environment interplay may underlie previously reported associations between environmental risk factors such as childhood adversity and psychotic outcomes. For instance, a twin-based study suggested that higher level of genetic risk associated with psychosis may moderate the impact of childhood adversity on the risk of adult psychotic symptom formation [31]. Furthermore, two recent studies provide evidence for gene-environment interaction effects in the association between psychosocial stress factors and European Psychiatry 28 (2013) 207-212 Purpose: To test whether the association between childhood adversity and positive and negative psychotic experiences is due to genetic confounding. Method: Childhood adversity and psychotic experiences were assessed in an ongoing sample of 226 twins from the general population. A monozygotic (MZ) twin differences approach was used to assess possible genetic confounding. Results: In the whole sample, childhood adversity was significantly associated with positive (b = 45; SE = 0.16; P = 0.008) and negative psychotic experiences (b = 0.77; SE = 0.18; P < 0.01). Within-pair MZ twin differences in exposure to childhood adversity were significantly associated with differences in positive (b = 71; SE = 0.29; P = 0.016) and negative psychotic experiences (b = 98; SE = 0.38; P = 0.014) in a subsample of 85 MZ twin pairs. Conclusions: Individuals exposed to childhood adversity are more likely to report psychotic experiences. Furthermore, our findings indicate that this association is not due to genetic confounding.
psychotic experiences in samples drawn from the general population [1,37]. However, it would be also important to clarify whether environmental factors per se have an impact on the expression of psychosis. So far associations between environmental risk factors and psychotic outcomes have been explored without controlling for genetic confounding [45], that is, individuals at increased genetic risk for psychosis may be more vulnerable to be victimised because of traits associated with psychosis, such as cognitive impairments, impaired social functioning, oddness or others. To the best of our knowledge, only one previous study provided evidence for an association between childhood trauma and risk to develop psychotic symptoms after controlling for genetic liability for psychosis [4]. Therefore, although childhood adversity as an environmental risk factor for psychosis has been extensively studied and the neurobiological impact of early adverse events in the brain is well-established [20,30,46], whether the association between childhood adversity and psychosis is likely causal or merely reflects gene-environment correlation remains to be examined. In this context, twin designs offer a unique opportunity to disentangle genetic and environmental effects on complex phenotypes such as psychotic experiences [9]. Specifically, the monozygotic (MZ) twin differences approach has been referred to as a strong test of the unique environmental experiences that make family members different from each other (also called non-shared environment) independently of genetics [12,32,47]. Since MZ twins are, nearly always, identical at the DNA sequence level [9]; phenotypic differences observed between MZ twins must be explained by differential exposure to environmental factors. In other words, if differences in the expression of subclinical psychotic experiences in MZ twins are associated with exposure to childhood adversity, this would provide strong evidence that the observed association between childhood adversity and psychosis is not due to genetic confounding. Therefore, the present study aimed to examine: whether childhood adversity was associated with positive and negative psychotic experiences in a twin sample from the general population; to what extent MZ twins were similar for their exposure to childhood adversity and presence of psychotic experiences; whether differences in exposure to childhood adversity were associated with differences in the expression of psychotic experiences in a subsample of MZ twins.

Participants
The sample consisted in 230 Spanish adult twins (115 twin pairs) from the general population, including 86 MZ twin pairs. The mean age was 34 years (SD = 13.28) and 34.2% of the subjects were males. Recruiting was conducted from the University of Barcelona Twin Register and media advertisements. The University of Barcelona Twin Register consists of a list of twin pairs from Catalonia who gave permission to be contacted for research purposes. Identified twin pairs were first contacted by telephone and invited to participate. A battery of psychological and neurocognitive tests was administered to the twins by trained psychologists (S.A. and X.G.). Of note, twins were requested to fill self-report questionnaires in separate rooms in order to avoid sharing of responses between twins and to ensure confidentiality. Twins were interviewed face-to-face for personal medical records (S.A. and X.G.). Also, lifetime DSM-IV-TR [2] Axis-I diagnosis was assessed in a face-to-face interview with a clinical trained clinical psychologist (X.G.) using the Structural Clinical Interview for DSM-IV disorders (SCID-I; [17]). Exclusion criteria applied were age under 17 and over 65 years, a medical history of neurological disturbance, presence of sensory or motor alterations and current substance misuse or dependence. All subjects were from Caucasian origin. Written informed consent was obtained from all participants after a detailed description of the study aims and design, approved by the local Ethics Committee.

Childhood adversity
To assess childhood adversity, we used an adapted version of the Adverse Childhood Experiences Questionnaire (ACEQ; [16]). This questionnaire assesses the exposure to events of childhood abuse, childhood neglect and household dysfunction. Three items regarding bullying and parental loss were added to the original version. Our adapted version consists on 19 items. Each item assesses the exposure to a particular adverse event. Participants are requested to answer ''yes'' or ''no'' to each item which indicates whether they were exposed or not to each adverse event. Items are detailed in the Appendix. All the positive answers are added up to obtain a total childhood adversity score which ranges from 0 to 19.

Psychotic experiences
The Community Assessment of Psychic Experiences (CAPE; [40]) was used to assess positive and negative psychotic experiences. This validated self-report questionnaire measures the lifetime prevalence of psychotic experiences in a frequency scale ranging from ''never'' to ''nearly always''. The positive dimension of the CAPE includes 20 items mainly referring to hallucinations and delusions such as ''do you ever feel as if things in magazines or TV were written especially for you?''. The negative dimension, which consists of 14 items, mainly assesses alogia, avolition, anhedonia and lack of interest in social relationships. An example of item is ''do you ever feel that you experience few or no emotions at important events?''. Participants are asked to indicate the frequency of ocurrence of psychotic experiences on a fourpoint scale (ranging from ''never'' to ''nearly always''). The instrument provides a total continuous score per dimension ranging from 20 to 80 in the positive dimension and from 14 to 56 in the negative dimension.

Zygosity
Zygosity was established genotyping 16 loci: 15 short tandem repeat (STR) loci and amelogenin, the gender determining marker. Genomic DNA was extracted from peripheral blood cells using the Real Extraction DNA Kit (Durviz S.L.U., Valencia, Spain). The PowerPlex 1 16 System (Promega Corporation) allowed the coamplification and three-color detection of 16 loci. Twins with only one divergent allele were genotyped a second time to limit the scope for genotyping error. Identity on all the markers can be used to assign monozygosity with greater than 99% accuracy [33].

Further characteristics of the sample
As data derived from this twin sample has not been published yet, further characteristics of the sample are reported for descriptive purposes. Apart from age and sex, sociodemographic characteristics include estimated Intelligence Quotient (IQ) assessed by four subtests (Block design, matrix reasoning, information and vocabulary) from the Wechsler Adult Intelligence Scale (WAIS-III; [48,36]); level of education (elementary school, high school and university), birth of place (''urban'' when the twins were born in the city of Barcelona and ''non-urban'' when they were born at other Spanish towns with lower number of habitants compared to Barcelona city) and socioeconomic status (SES). A continous score representing SES was obtained using four-factor index of social status developed by Hollingshead [15,23]. SES scores ranging from 8 to 30 were defined as ''Low SES'' and scores between 31 and 66 were classified as ''Average SES'' [13]. Of note, some of these measures were not available for all subjects.

Statistical analysis
Data was analysed in three phases. First, multiple regression models were conducted to map the association between childhood adversity and positive and negative psychotic experiences. In these models, childhood adversity was the variable of interest, sex and age were included as covariates and positive and negative psychotic experiences were used as the outcome measures. Separate models were conducted for each outcome measure. The non-independence of clustered twin data was corrected for by using tests based on the sandwhich or Huber/White variance estimator [51]. In these analyses, the individual was the unit of analysis.
Second, MZ intrapair correlations were calculated for childhood adversity and positive and negative psychotic experiences. These analyses let us confirm that MZ twins differed in their exposure to childhood adversity and their scores for positive and negative psychotic experiences. The proportion of the variance of the phenotype which can be directly attributable to unique environment (which includes measurement error) can be obtained by this formula: 1 -rMZ, where r represents the within-pair correlation [34].
Third, associations between intrapair differences in childhood adversity, positive and negative psychotic experiences were analysed by linear regression analysis. Intrapair scores were calculated for childhood adversity, positive and negative psychotic experiences by substracting the score of the Twin 2 from the score of Twin 1 (Twin 1-Twin 2). Twins were randomly assigned to be 1 or 2. Associations between intrapair differences in CA and intrapair differences in positive and negative PEs scores were conducted in a subsample of 85 MZ twin pairs. Because intrapair analyses in MZ twins fully control for genetic influences, any association between the abovementioned variables would be attributable to environmental factors [32,35] and thus, reject the hypothesis that the association is due to genetic confounding. In the last two analyses each MZ twin pair was the unit of analysis.
Statistical analyses were carried out in STATA 10.0 [39] following the procedures described in Carlin et al. [11].

Results
Most of the sample was composed of females (66.1%), the average age was 33.8 years (SD = 13.3) and more than half of the sample had completed university educational level (59.3%). Average IQ scores were within the normal range for non-clinical samples (103.3; SD = 11.5). Around half of the sample was born in non-urban areas (58.7%). Most of the twins were of average SES level (65.8%). The MZ twin subsample showed very similar sociodemographic characteristics compared to the whole twin sample (Table 1).
In the whole sample, positive CAPE score ranged from 13 to 39 (mean = 25.3; SD = 4.0) and negative CAPE score ranged from 12 to 49 (Mean = 22.1; SD = 4.8). CAPE scores were very similar in the MZ twins subsample (CAPE positive: Mean = 25.7; SD = 4.24; CAPE Negative: Mean = 22.5; SD = 5.05). In order to obtain the prevalence of psychotic experiences in the current sample, CAPE scores were recoded to 0 (never, sometimes) and 1 (often, almost always). Specifically, 37.1 to 38.8% of the sample often, or almost always, experienced at least one positive or negative psychotic experience. Similarly, in the MZ twin subsample, 41% of the sample often, or almost always, experienced at least one positive or negative psychotic experience.
With regard to childhood adversity (CA) score, the mean was 2.0 (SD = 2.2) and it ranged from 0 to 14. In the MZ twin subsample, CA score also ranged from 0 to 14 and the mean was 2.0 (SD = 2.4). In the whole sample, 26.3% of the individuals did not experience any adverse childhood event and 26.3% reported one adverse childhood event; the rest of the sample reported two or more adverse childhood events. Similarly, in the MZ twin subsample, 27.3% of the individuals did not report any adverse childhood event, 26% reported one adverse childhood event and the rest reported two or more adverse childhood events.
First, regarding the association between CA and psychotic experiences, analyses based on the whole sample showed that CA was significantly associated with both positive (b = 0.45; SE = 0.16; P = 0.008) and negative psychotic experiences (b = 0.77; SE = 0.18; P < 0.01) ( Table 1). These analyses were adjusted for clustering.
Second, we conducted within-pair correlations to index the similarity for the outcome measures and the variable of interest between twin 1 and twin 2 in the subsample of MZ twin pairs. The within-pair correlations for positive and negative psychotic experiences were r = 0.48 (P < 0.01) and r = 0.44 (P < 0.01) respectively. Therefore, around 52 to 56% of the variance of CAPE could be attributed to unique environmental factors not shared by twins. In regard to CA score, the within-pair correlation was r = 0.79 (P < 0.01). Thus, although most of the childhood adverse events experienced by the twins are common, some of them are specific (21% of the variance of childhood adversity). Finally, associations between intrapair differences in childhood adversity, positive and negative psychotic experiences were analysed. The mean score of within-pair differences for childhood adversity, positive and negative psychotic experiences was 0.13 (SD = 1.6; range = -4 -4), -0.17 (SD = 4.1; range = -15 -10) and-0.41 (SD = 5.3; range = -30-12) respectively. Regression analyses using within-pair MZ differences showed that MZ differential exposure to childhood adversity was significantly related to phenotypic differences in both positive (b = 66; SE = 0.28; P = 0.026) and negative dimensions of psychotic experiences (b = 93; SE = 0.37; P = 0.014) ( Table 2).

Discussion
To our knowledge, this is the first study adding evidence to the growing literature on the relationship between childhood adversity and psychotic experiences using an MZ-twin differences approach. The MZ-twin differences design ensures that pure unique or non-shared environmental effects, rather than geneenvironment interaction or evocative gene-environment correlation, are quantified [47].
Firstly, the present twin sample from the general population showed similar means and prevalences of psychotic experiences to those reported previously in singleton samples [6,1]. A recent study demonstrated a significant impact of the type of instrument used on the rate of psychotic experiences that was found [29]. Therefore, it is worth mentioning that we are comparing our means and prevalences obtained for CAPE scores with studies which used the same instrument [6,1].
With respect to childhood adversity, in a large communitybased study using the original version of the ACE questionnaire, 36.1% of the sample reported 0 adverse childhood experiences, 26.0% reported one adverse childhood experience and the rest reported two or more adverse childhood experiences [3]. These prevalences are very similar to those reported in the current sample.
Secondly, in agreement with previous studies [27,38,24,50], our findings provide support for the association between childhood adversity and psychotic experiences in the general population.
Thirdly, the fact that within-pair MZ correlations were not equal to 1 for any of the measured phenotypes indicated that we could test for unique environmental effects of childhood adversity on psychotic experiences.
Fourthly, regarding the primary goal of the current study, within-pair MZ differences in exposure to childhood adversity were significantly related to phenotypic differences for both positive and negative dimensions of psychotic experiences. Because the members of the MZ twin pair are genetically identical to each other, any environmental effects operate upon genotype effects that do not differ between the members of the MZ twin pair [47]. These findings indicate that the association between childhood adversity and psychosis cannot be solely attributed to genetic confounding and thus, that childhood adversity may represent a true risk factor for the development of psychotic experiences.
These results are in agreement with those reported by Arseneualt et al. [4], who reported that childhood adversity may constitute a risk factor for the development of psychotic symptoms independently of the genetic background of the individual.
Proposed neurobiological and psychological mechanisms of risk underlying this association also add plausibility to these findings. Converging evidence from neurobiology and epidemiology suggests that early adverse events cause enduring brain dysfunction [20,10,21]. Persistent exposure or impact of stressors in the developing brain has been proposed to lead to chronically heightened stress-induced glucocorticoid release which, in turn, may impact on the hypothalamic-pituitary-adrenal (HPA) axis. Dysregulation of the HPA axis has been suggested to contribute to the dopaminergic abnormalities that are generally thought to be involved in the expression of psychotic phenotype [46,28]. At the psychological level, exposure to early adversity may create, also, a cognitive vulnerability, characterized by a tendency to perceive the self as powerless and others as malevolent, which in combination with an externalizing attribution style may ultimately lead to paranoid interpretation of anomalous experiences [5,19]. These risk mechanisms could be moderated by genetic variants, making some individuals more sensitive to psychosocial stress factors than others [1,37].
The results of the present study should be interpreted in the context of its limitations. First, due to the limited sample size our findings require replication and have to be interpreted with caution. Also, most of the sample consists of women and this may limit generalization of our findings. However, the sample showed to be representative of the general population regarding the sociodemographic characteristics and the prevalences of the variables studied and all the analyses were adjusted by sex. Second, the cross-sectional nature of our design did not allow inference of causal associations. Third, the retrospective measure of CA may be influenced by recall bias. Nevertheless, it is worth it to mention that retrospective self-reports of childhood trauma are more likely to be an underestimation of the true prevalence of childhood maltreatment than an overestimation [22]. Fourth, it has been shown that the impact of childhood adversity on psychosis may depend on the type or frequency of such events [18]. However, there was insufficient power to investigate the impact of unique environmental effects of specific types of childhood adversity or reporting one versus reporting multiple childhood adverse events in the present sample of MZ twins.
Finally, as other studies using an MZ-twin differences design [12,47], we cannot rule out the possibility that some unmeasured non-genetic factor could have contributed to our findings. Table 2 In the left side of the table, association between Childhood Adversity (CA) score and positive and negative psychotic experiences (PEs) in the whole sample (n = 226) adjusting for the non-independence nature of the data. In the right side of the table, association between intrapair scores (twin 1-twin 2) for CA and intrapair scores for positive and negative PEs in a subsample of MZ twin pairs (n = 85 pairs). All analyses were adjusted by sex and age. Therefore, further research in larger samples is needed to better understand under which circumstances childhood adversity environmentally increases the risk or frequency of psychotic experiences.

Conclusion
Our findings shed new light regarding the role of childhood adversity as an environmental risk factor involved in the development of psychotic experiences. We found a significant environmental effect of childhood adversity on the development of positive and negative psychotic experiences using an MZ-twin differences approach, suggesting that the association cannot be solely attributed to genetic confounding. Therefore, although some individuals may be genetically vulnerable to the impact of childhood adversity [1], our findings indicate that childhood adversity can independently contribute to the development of psychotic experiences. Further research is needed to better understand under which circumstances childhood adversity environmentally increases the risk or frequency of psychotic experiences.

Disclosure of interest
The authors declare that they have no conflicts of interest concerning this article.