Effects of copy number variants in schizophrenia on longitudinal psychosocial functioning Katrin Gade* 1,7 , Urs Heilbronner* 1 , Franziska Degenhardt 2,5 , Jana Strohmaier 3 , Maren Lang 3 , Josef Frank 3 , Jens Treutlein 3 , Anna Koller 5 , Andrea Hofmann 5 , Stephanie H. Wi 3 , Sven Cichon 2,5,6 , Markus M. Nöthen 2,5 , Marcella Rietschel 3 , Thomas G. Schulze 1,3,7 *ese authors contributed equally 1 Institute of Psychiatric Phenomics and Genomics, Ludwig-Maximilians-University Munich, Germany. 2 Institute of Human Genetics, University of Bonn, Germany, 3 Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Mannheim, Germany, 4 Institute of Neuroscience and Medicine (INM-1), Research Centre Jülich (FZJ), Jülich, Germany, 5 Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Germany, 6 Division of Medical Genetics, University Hospital Basel, University of Basel, Switzerland, 7 Department of Psychiatry and Psychotherapy, University Medical Center, Georg-August-University, Göttingen, Germany Abstract Individual illness severity may be measured by the degree of overall psychosocial funconing. We studied whether the presence of one or more copy number variants (CNVs) is associated with the level of psychosocial impairment measured by the Global Assessment of Funconing (GAF; DSM- IV Axis V) scale in a sample of individuals with DSM-IV schizophrenia (SZ). The GAF score measures the overall funconing level of an individual from 1 (lowest) to 100 (highest). Using a genome-wide, high-quality CNV dataset, we assessed whether CNVs are related to GAF values collected for three points in me over the individual course of disease: before illness onset, the “worst ever” (during an illness episode) and the current (in remission) GAF score. Invesgang GAF values adjusted for phenotypic predictors, our analysis revealed a trend towards lower psychosocial funconing at the “worst ever” GAF in individuals possessing one or more CNVs compared to individuals without CNVs. An exploratory analysis of CNVs present in the study sample found a protecve effect on the current GAF score for a duplicaon on chromosome 10q26.3. Introduction Psychosocial impairment, common to all mental disorders, is especially pronounced in SZ. CNVs have been shown to affect vulnerability to disease (e.g. Szatkiewicz et al., 2014) and certain CNVs previously linked to SZ have been shown to affect cognion and GAF scores also in individuals without SZ (Stefans- son et al., 2014). Therefore, we hypothesized CNV load to contribute to the degree of psychosocial im- pairment also within SZ individuals. To invesgate this issue, we studied a sample of SZ paents for whom the levels of psychosocial funconing at mulple points in me over the course of the disorder had been characterized. Methods CNV data We took advantage of a large (n=1,637) exisng genome-wide CNV data set of paents with a DSM-IV diagnosis of schizophrenia or schizoaffecve disorder (Priebe et al., 2013) to idenfy deleons and du- plicaons. All individuals were genotyped on Illumina HH550/H610Q/H660W (lllumina, San Diego, USA) whole-genome SNP arrays, fulfilling standard quality control criteria. Detailed informaon on detecon and quality control is provided elsewhere (Degenhardt et al., 2012). CNVs were selected based on the fol- lowing criteria: 1. detecon by PennCNV (Wang et al., 2007) and QuanSNP (Colella et al., 2007) soſtware, 2. ≥30 covered SNPs, 3. confidence value/log Bayes Factor ≥30 and 4. overlapping with RefSeq genes. We excluded all regions reported by Levinson et al. (2011), as these are prone to false-posives (e.g. telomeric and centromeric regions). All CNVs were visually inspected in GenomeStudio (lllumina, San Diego, USA). GAF data We used the Global Assessment of Funconing (GAF; DSM-IV Axis V) scale to assess psychosocial funconing. For 266 individuals, informaon on GAF was available for three points in me (see below). We used linear regressi- on to adjust psychosocial funconing levels for age at onset/duraon of di- sease and sex, major phenotypic con- tributers to psychosocial funconing (Gade et al., 2015). We analyzed the premorbid GAF, the “worst ever” GAF (during an illness episode) and the cur - rent (in remission) GAF. References Colella S. et al. (2007) Nucleic Acids Res 35:2013-25. Degenhardt F. et al. (2012) Am J Med Genet B Neuropsychiatr Genet 159B:263-73. Gade K. et al. (2015) World J Biol. Psychiatry 16:237-48. Levinson D.F. et al. (2011) Am J Psychiatry 168:302-16. Malzahn D. et al. (2010) Genet Epidemiol 34:469-78. Priebe L. et al. (2013) PLoS One 8:e64035. Rees E. et al. (2014) Br J Psychiatry. 204:108-14. Stefansson H. et al. (2014) Nature 505:361-6. Szatkiewicz J.L. et al. (2014) Mol Psychiatry 19:762–73. Wang K. et al. (2007) Genome Res 17:1665-74. Wichen H.-U., Fydrich T. (1997) Strukturiertes Klinisches Interview für DSM-IV (SKID-I und SKID-II). Hogrefe. −20 −10 0 10 20 30 40 No CNVs CNVs Worst ever GAF residuals p=0.11 Figure 1. The presence of one or more CNVs is associated with lower psychosocial funconing at the “worst ever” GAF (adjusted for duraon of illness and sex) in SZ paents. Horizontal lines show medians of adjusted GAF scores. Figure 2. A duplicaon on chromosome 10q26.3 has protecve effects on current GAF scores (adjusted for duraon of illness and sex). Visualizaon of CNVs in UCSC Genome Browser using NCBI36/hg18 as genome reference. Results CNV load and psychosocial funconing Table 1 displays descripve data of our sample and raw GAF scores. Analyses using Wilcoxon tests revea- led a trend towards lower psychosocial funconing at the “worst ever” GAF in paents with larger CNVs (>30 SNPs; Figure 1). All other measures of psychosocial funconing were not affected by the CNVs ana- lyzed. The non-parametric longitudinal test did not detect any effect of CNVs on GAF values either. Exploratory analysis of CNVs present in our study sample Regarding specific CNVs (see Table 2), Wilcoxon tests showed a protecve effect of duplicaons on chro- mosome 10q26.3 (Figure 2) on current GAF scores. This region encompasses the genes SCART1 (scaven- ger receptor protein family member; non-coding RNA), CYP2E1 (cytochrome P450, family 2, subfamily E, polypepde 1) and SYCE1 (synaptonemal complex central element protein 1, transcript variant 1). CNVs in other invesgated regions, including those previously associated with SZ, did not influence GAF scores. Discussion This is the first analysis relang CNVs to psychosocial funconing. We provide tentave evidence for an effect of CNVs on psychosocial funconing within SZ paents. In line with their effect in non-diseased in- dividuals (Stefansson et al., 2014), CNVs appear to have negave influence on psychosocial funconing also within SZ paents. The analysis of specific CNVs present in our study sample found suggesve evidence for a protecve ef- fect of a duplicaon in a region containing the genes SCART1 (non-coding RNA), CYP2E1 and SYCE1. This implicates non-coding RNA, possibly via an effect on transcripon, drug metabolism and/or molecules involved in meiosis in the pathway between genecs and psychosocial funconing. Due to the small sample size, invesgaon in a larger independent sample is warranted to confirm our at best nominally significant results. Also, while the use of high-quality CNVs may be required to avoid false-posive findings, this approach may also have impeded the detecon of true effects. Also, we only focused on large CNVs whereas effects of smaller CNVs remain to be invesgated. Acknowledgements / Financial Disclosure We thank all of the paents for parcipang in this study. The study was funded by the German Fe- deral Ministry of Educaon and Research (BMBF) through the Integrated Genome Research Network (IG) MooDS (Systemac Invesgaon of the Molecular Causes of Major Mood Disorders and Schizo- phrenia; grant 01GS08144 to MMN and SC, grant 01GS08147 to MR) under the auspices of the Na- onal Genome Research Network plus (NGFNplus), and through the Integrated Network IntegraMent (Integrated Understanding of Causes and Mechanisms in Mental Disorders), under the auspices of the e:Med Program. MMN received support from the Alfried Krupp von Bohlen und Halbach-Sf- tung and is a member of the Deutsche Forschungsgemeinschaſt (DFG)-funded Excellence Cluster Im- munoSensaon. MR was also supported by the 7th Framework Programme of the European Union (ADAMS project, HEALTH-F4-2009-242257; CRESTAR project, HEALTH-2011-1.1-2) grant 279227. FD received support from the BONFOR Programme of the University of Bonn, Germany. TGS received funding from DFG (Klinische Forschergruppe 241: SCHU 1603/5-1) and by the Lisa-Oehler-Founda- on. These funding sources had no involvement in the study design; the collecon, analysis, and in- terpretaon of data. N 266 Age at interview 36.4±0.69 Female 42% Age at onset 26.1±0.55 Duraon of illness 10.3±0.61 Premorbid GAF 84.5±0.65 “Worst ever” GAF 25.9±0.55 Current GAF 63.2±0.93 No. CNVs (range) per ind. 0.64 (0-4) No. DUPs (range) per ind. 0.52 (0-4) No. DELs (range) per ind. 0.13 (0-2) Table 1. Characteriscs of the SZ sample. All data are reported as mean±SEM if not specified otherwise. Abbreviaons: DEL – deleon, DUP – duplicaon. Locaon DEL/DUP GAF1 GAF2 GAF3 p (95%CI) p (95%CI) p (95%CI) 6:31468368-31559455 0/3 0.92 (-15.0,12.7) 0.36 (-8.2,11.4) 0.48 (-12.3,23.6) 6:162637688-162834976 1/2 0.31 (-7.5,21.3) 0.24 (-1.2,10.4) 0.69 (-24.7,15.9) 6:168071512-16839100 0/6 0.26 (-4.5,14.2) 0.89 (-4.7,5.2) 0.85 (-14.9,12.2) 10:47013328-47173619 0/22 0.86 (-4.2,3.5) 0.82 (-2.4,2.8) 0.12 (-1.2,11.0) 10:67987089-68281109 1/2 0.98 (-14.5,18.9) 0.62 (-13.9,8.9) 0.18 (-6.3,25.0) 10:135116379-135227438 0/13 0.14 (-9.2,1.1) 0.74 (-3.5,5.3) 0.05 (-16.0,-0.08) 22:15434720-1584185 0/4 0.15 (-3.2,22.5) 0.34 (-10.8,11.7) 0.18 (-6.2,28.9) 22:23994408-24240667 5/11 0.1652 (-7.3,1.3) 0.45 (-5.1,3.6) 0.47 (-10.1,4.9) SZ associated regions 4/4 NS (NA) NS (NA) NS (NA) (1q21.1, 2p16.3, 15q11.2, 16p13.11, 16p11.2) Table 2. Exploratory analysis of specific CNVs present in the study sample. GAF scores adjusted for phenotypic predictors were compared using non-pa- rametric Wilcoxon tests. All CNVs present in more than three individuals were tested. NA – not applicable, NS – not significant. Subjects Diagnoses were established by the German version of the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I; Wichen and Fydrich, 1997). All paents were of German descent according to self-reported ancestry and were recruited from consecuve hospital admissions at the Central Instute of Mental Health, Mannheim, and the Department of Psychiatry, University of Bonn, Germany. The study protocol was approved by the local ethics commiees and informed wrien consent was obtained prior to study parcipaon from all parcipants. Stascal analyses We used Wilcoxon non-parametric rank tests to compare psychosocial funconing between paents carrying one or more CNVs with paents carrying no CNVs. We also looked for possible longitudinal ef- fects of CNVs using the longitudinal non-parametric test (LNPT; Malzahn et al., 2010). Furthermore, we explored the effects of CNVs in specific chromosomal regions on GAF scores. We invesgated all regions with CNVs present in at least three individuals in our sample and, independently, also assessed CNV re- gions previously associated with SZ (Rees et al., 2014). Conflict of Interest There are no conflicts of interest.