Association of Genetic Liability to Psychotic Experiences With Neuropsychotic Disorders and Traits

Key Points Question Is the genetic liability to psychotic experiences shared with schizophrenia and/or other neuropsychiatric disorders and traits? Findings In this cohort study, genetic correlation, polygenic risk score, and copy number variation analyses indicated a shared genetic liability between psychotic experiences and major depressive disorder, schizophrenia, bipolar disorder, and neurodevelopmental disorders. Genome-wide association studies identified 4 genetic loci associated with psychotic experiences including loci in ANK3 and CNR2. Meaning Findings suggest that the genetic liability of psychotic experiences is shared with several psychiatric disorders, which include, but is not specific to, schizophrenia.


eAppendix. Unusual and Psychotic Experience Questions in UK Biobank
The following questions are from Section F of the Mental Health Questionnaire (MHQ) administered via web-based questionnaire for UK Biobank participants.

Q. No Question Responses
INTRO The next set of questions is about unusual experiences that you may have had, like seeing visions or hearing voices. We believe that these things may be quite common, but we don't know for sure. So please take your time and think carefully before answering. F1 Did you ever see something that wasn't really there that other people could not see?
Please do not include any times when you were dreaming or half-asleep or under the influence of alcohol or drugs.
[Choose one from] -01 Yes -00 No -NA Do not know -DA Prefer not to answer F1a About how many times in your life did this happen (when you were not dreaming, not half-asleep, and not under the influence of alcohol or drugs)?
FBOX1: Integer box 1 -999 FBOX1 & "time(s)" OR -01 Too many to count -NA Do not know -DA Prefer not to answer F2 Did you ever hear things that other people said did not exist, like strange voices coming from inside your head talking to you or about you, or voices coming out of the air when there was no one around?
Please do not include any times when you were dreaming or half-asleep or under the influence of alcohol or drugs.
[Choose one from] -01 Yes -00 No -DA Prefer not to say -NA Don't know

F2a
About how many times in your life did this happen (when you were not dreaming, not half-asleep, and not under the influence of alcohol or drugs)?
FBOX2: Integer box 1 -999 FBOX2 & "time(s)" OR -01 Too many to count -NA Do not know -DA Prefer not to answer F3 Did you ever believe that a strange force was trying to communicate directly with you by sending special signs or signals that you could understand but that no one else could understand (for example through the radio or television)?
Please do not include any times when you were dreaming or half-asleep or under the influence of alcohol or drugs.
[Choose one from] -01 Yes -00 No -NA Do not know -DA Prefer not to answer

F3a
About how many times in your life did this happen (when you were not dreaming, not half-asleep, and not under the influence of alcohol or drugs)?
FBOX3: Integer box 1 -999 FBOX3 & "time(s)" OR -01 Too many to count -NA Do not know -DA Prefer not to answer F4 Did you ever believe that that there was an unjust plot going on to harm you or to have people follow you, and which your family and friends did not believe existed?
Please do not include any times when you were dreaming or half-asleep or under the influence of alcohol or drugs.
[Choose one from] -01 Yes -00 No -NA Do not know -DA Prefer not to answer F4a About how many times in your life did this happen (when you were not dreaming, not half-asleep, and not under the influence of alcohol or drugs)?
FBOX4: Integer box 1 -999 FBOX4 & "time(s)" OR -01 Too many to count -NA Do not know -DA Prefer not to answer F5 How often did any of these experiences happen in the past 1 year (seeing a vision, hearing a voice, or believing that something strange was trying to communicate with you, or there was a plot against you)?
[Choose one from] -00 Not at all -01 Once or twice -02 Less than once a month -03 More than once a month -04 Nearly every day or daily -DA Prefer not to answer F6 How old were you (approximately) when you first had one of these experiences (seeing a vision, hearing a voice, or believing that something strange was trying to communicate with you, or there was a plot against you)?
FBOX5: Integer box 2 to current age FBOX5 & "years old" OR -01 As long as I can remember -NA Do not know -DA Prefer not to answer F7 How distressing did you find having any of these experiences (seeing a vision, hearing a voice, or believing that something strange was trying to communicate with you, or there was a plot against you)?
[Choose one from] -00 Not distressing at all, it was a positive experience -01 Not distressing, a neutral experience -02 A bit distressing -03 Quite distressing -04 Very distressing -NA Do not know -DA Prefer not to answer F8 Did you ever talk to a doctor, counselor, psychiatrist or other health professional about any of these experiences (seeing a vision, hearing a voice, or believing that something strange was trying to communicate with you, or there was a plot against you)?
[Choose one from] -01 Yes -00 No -NA Do not know -DA Prefer not to answer F9 Were you ever prescribed a medication by a health professional for any of these experiences (seeing a vision, hearing a voice, or believing that something strange was trying to communicate with you, or there was a plot against you)?

eMethods 2. Defining European Genetic Ancestry
Analyses were restricted to individuals with a self-reported British and Irish ethnic background (UK Biobank field ID: 21000). The first 40 principal components supplied by UK Biobank 1 (UK Biobank field ID: 22009) were used to assess and control for population structure. Initial analyses (eFigure 2) showed that filtering by self-reported ancestry does not adequately control for ancestry, and so European genetic ancestry was confirmed using the 'covMCD' function in the R package 'robustbase' 2 , which uses the first five principal components to compute a Minimum Covariance Determinant (MCD) estimator of location and scatter via the deterministic MCD algorithm 3 . The MCD defines a hyper-ellipsoid in a multi-dimensional space that contains the majority of points representing individuals in that group, and once this has been defined, all individuals are allocated a distance to the hyper-ellipsoid in PC space 4 . We selected individuals that were within the 90 th percentile of MCD distance (eFigure 3).

eMethods 3. Replication in ALSPAC Cohort
To assess the validity of the psychotic experience phenotype, we targeted psychotic experiences in the Avon Longitudinal Study of Parents and Children (ALSPAC) longitudinal birth cohort.

ALSPAC study population
The initial cohort consisted of 14,541 pregnant women residing in the former Avon Health Authority area with an expected delivery date between April 1991 and December 1992 5,6 . Of these initial pregnancies, there was a total of 14,676 fetuses, resulting in 14,062 live births and 13,988 children who were alive at 1 year of age. When the oldest children were approximately 7 years of age, an attempt was made to bolster the initial sample with eligible cases who had failed to join the study originally. The total sample size for analyses using any data collected after the age of 7 is therefore 15

ALSPAC genetic data
Genetic data were acquired using the Illumina HumanHap550 quad genome-wide single nucleotide polymorphism (SNP) genotyping platform from 9912 participants. Individuals were excluded from further analysis based on gender mismatches, minimal or excessive heterozygosity, disproportionate levels of individual missingness (>3%), evidence of cryptic relatedness (>10% of alleles identical by descent) and being of non-European ancestry (assessed by multidimensional scaling analysis including HapMap 2 individuals). SNPs with a minor allele frequency (MAF) of < 1%, Impute2 information quality metric of < 0.8, a call rate of < 95% or evidence for violations of Hardy-Weinberg equilibrium (pvalue < 5 x 10 -7 ) were removed. Imputation of the target data was performed using Impute v2.2.2 against the 1000 genomes reference panel (Phase 1, Version 3; all polymorphic SNPs excluding singletons), using 2,186 reference haplotypes (including non-Europeans). Following quality control assessment and imputation and restricting to 1 young person per family, genetic data was available for 7975 ALSPAC individuals.

Generating psychotic experiences polygenic risk scores in ALSPAC
Prior to construction of polygenic risk scores (PRSs) for psychotic experiences, SNPs were removed from the analysis if they had a MAF < 0.01, an imputation quality < 0.8, or if there was allelic mismatch between samples (the alleles reported by the discovery study did not match the alleles present in the ALSPAC sample). Due to the high linkage disequilibrium (LD) within the extended major histocompatibility complex (MHC; chromosome 6: 25-34Mb) only a single SNP was included to represent this region within the analysis. Remaining SNPs were then further pruned for LD using the PLINK (v1.90) 7 --clump command to retain SNPs with an association p-value ≤ 0.5 and r2 < 0.25 within 500kb windows.
PRSs were constructed using the summary statistics from the primary GWAS of any psychotic experience in UK Biobank. PRSs were calculated for each ALSPAC individual using the PLINK (v1.07) 7 --score command. Scores are calculated by summing the number of reference alleles present for each SNP (0, 1 or 2) weighted by the logarithm of its odds ratio for any psychotic experience and standardized prior to analyses.
Our primary analysis used a score generated using a list of SNPs meeting an any psychotic experience GWAS p-value threshold of ≤ 0.50. To assess the robustness of our findings, analyses were then repeated using PRSs based on SNPs that were associated with any psychotic experience at a range of GWAS p-value thresholds (p-value ≤ 1e -6 to ≤ 0.5).

Psychotic experiences in ALSPAC
The semi-structured Psychosis-Like Symptom Interview (PLIKSi) 8,9 , which draws on principles of standardized clinical examination developed for the Schedule for Clinical Assessment in Neuropsychiatry (SCAN), was used to assess psychotic experiences in ALSPAC at ages 12 and 18 years. The PLIKSi allows rating of 12 psychotic experiences including hallucinations (visual and auditory), delusions (spied on, persecution, thoughts read, reference, control, grandiosity, other) and experiences of thought interference (broadcasting, insertion and withdrawal). Any unspecified delusions elicited are also rated. Structured stem questions (e.g. "have you ever seen something or someone that other people could not see?"; "have you ever thought you were being followed or spied on?"; "Have you ever felt that thoughts are put into your mind that are not your own?") are followed up by cross-questioning to allow the interviewer to make a decision as to whether experiences described meet SCAN criteria for a psychotic experience.
The interviewers were psychology graduates trained in assessment using the SCAN psychosis section and using the PLIKSi. Psychotic experiences were rated as not present, suspected or definitely psychotic. Unclear responses were always 'rated down' and symptoms were rated as definite only when a clear example was provided. At regular intervals samples of recorded interviews were also rated by a psychiatrist to ensure interviewers were rating experiences correctly. The PLIKSi shows very good inter-rater and test-retest reliability 10 .
To maximise the numbers within our sample we used data from both the interviews at ages 12 and 18 years to identify individuals rated as having one or more definite psychotic experiences between ages 12 and 18 years, compared to no or only suspected psychotic experiences across this age range.
To assess the sensitivity of results, analyses were also performed using data from age 12 and age 18 years separately and using a different cut-off of definite or suspected psychotic experiences compared to no psychotic experiences at 12 or 18 years. A latent variable was also generated for psychotic experiences at age 16.5 years using ten items from the self-report Psychosis-Like Symptoms Questionnaire (PLIKSq) 11 . These ten items were rated on a 3-point scale (never; maybe; definitely) and assessed presence of hallucinations, delusions and thought interference since age 15. The psychotic experience latent variable was generated in Mplus (version 7.31) as previously reported 12 .
Logistic regressions were performed in Stata v15.1 to test for associations between any psychotic experience PRSs and ALSPAC psychotic experiences reported at age 12 and 18 years. Linear regressions were used in Mplus to test for associations between any psychotic experience PRSs and the latent psychotic experience trait at age 16.5 years. No principal components were included in the PRS regression analyses, as is standard for studies in this cohort due to homogenous nature of the sample 13 .

eMethods 4. Calculation of Polygenic Risk Scores in UK Biobank
To examine the relationship between psychotic experiences and genetic risk for schizophrenia, bipolar disorder, depression, attention deficit/hyperactivity disorder (ADHD), autism spectrum disorder, neuroticism and intelligence, PRSs were generated using the method described by the PGC 14 . External discovery datasets used to generate the PRSs included; schizophrenia 15 , bipolar disorder 16 , major depressive disorder 17 , ADHD 18 , autism spectrum disorder 19 , neuroticism 20 , and lastly for intelligence, summary statistics were obtained for the latest GWAS 21 excluding UK Biobank participants (n=74,214 individuals remaining, summary statistics specifically derived for this study). PRSs were calculated using imputation dosage data for each UK Biobank participant that passed GWAS QC measures using PRSice (v2) 22 . High quality SNPs were selected by applying filters for INFO > 0.9, MAF > 0.1, removing indels, and excluding the extended MHC region (25 Mb -35 Mb). A reference panel of 1000 randomly selected UK Biobank participants was used to obtain relatively independent SNPs (r 2 < 0.2, window size < 500kb). The first five principal components, any additional principal components from the first 20 that were nominally associated (p < 0.05) with the GWAS phenotype in a logistic regression, and genotyping array, were added as covariates for each PRS generated. Scores were generated at 11 SNP p-value thresholds (5 x 10 -8 , 1 x 10 -7 , 5 x 10 -6 , 5 x 10 -5 , 5 x 10 -4 , 0.005, 0.05, 0.10, 0.20, 0.50, 1).
Our primary analysis of PRS used standardised scores generated from SNPs with a GWAS discovery sample p-value threshold of p ≤ 0.05. Statistical association analyses were conducted in R and used a regression model to test the association of each PRS with the various psychotic experience phenotypes, and covarying for the first five principal components and genotyping array. The Nagelkerke R 2 and area under the curve (AUC) values were adjusted for covariates.

eMethods 5. CNV Calling
CNV calling, which has been described in detail elsewhere 23 , was carried out using biallelic markers common to both genotyping platforms using PennCNV-Affy 24 . Exclusion criteria included ≥ 30 CNVs, waviness factor <-0.03 or > 0.03, call rate < 0.96 for individual samples, LRR SD > 0.35 and coverage of < 20 probes, density coverage of < 1 probe per 20,000 base pairs or a confidence score of < 10 for individual CNVs.
We compared carrier status of rare CNVs previously associated with (i) schizophrenia 25 and (ii) neurodevelopmental disorders more widely 26 (which includes all schizophreniaassociated CNVs and excludes 15q11.2 duplication because of its high frequency following our previous publication 23 ) with the three primary psychotic experience phenotypes used for GWAS. Association analyses were carried out using logistic regression and included age, sex and genotyping array as covariates.

eMethods 6. Individuals Excluded
The Quality control step 1 consisted of excluding study individuals that (i) did not have a selfreported White British or Irish ethnicity, (ii) did not have genetic data available, or (iii) did not pass initial genetic quality control parameters (missingness).
Step 2 consisted of excluding individuals that did not have European genetic ancestry as defined by principal components (see 'defining European genetic ancestry' above).
Step 3 consisted of excluding related individuals and finally, step 4 excluded individuals with a schizophrenia, bipolar disorder or psychotic disorder diagnosis.

eFigure 1. Psychotic Experience Phenotypes
Venn diagram detailing the sample overlaps between the phenotypes used in the three psychotic experience GWAS analyses. The left hand circle represents the comparison group, who reported no psychotic experiences. The right hand circle represents the cases used in the three GWAS (i) any psychotic experience, (ii) distressing psychotic experiences, and (iii) multiple occurrences of psychotic experiences.   . The x-axis shows the psychotic experience phenotype (any psychotic experience, distressing psychotic experiences and multiple occurrence psychotic experiences). Points display the odds ratio (OR) and error bars represent the 95% confidence intervals for the OR. Validation of psychotic experiences GWAS in ALSPAC cohort. Columns represent age at which psychotic experience measure is based, the psychotic experience measure targeted, the PRS training p-value threshold for SNP inclusion, effect size (see below notes), standard error (SE), p-value, lower 95% confidence interval of the effect size, upper 95% confidence interval of effect size, pseudo r2 and total number of individuals included (N). † Note that for PEs measured at 12 and 18 years, effect sizes represent odds ratios per standard deviation increase in PRS. For the PE latent trait at age 16.5 years effect sizes represent standard deviation change in latent trait per standard deviation change in PRS.