Copy number variation plays a clear role in the etiology of many psychiatric disorders, particularly schizophrenia. We performed array-CGH to look for copy number variants between five pairs of monozygotic twins discordant for bipolar disorder or schizophrenia. Our study found no differences in copy number variants between the sets of twins. Although alluring, realistic accounting for heterogeneity and chimerism highlight the technological limitations in studying monozygotic twins discordant for psychiatric disorders

Bloom, Rachael J.

Cannon, Tyrone D.

Chen, Guanhua

Collins, Ann L.

Hultman, Christina

Kähler, Anna K.

Sullivan, Patrick F.

PubMed

Carolina Digital Repository

Comprehensive Analysis of Copy Number Variation inMonozygotic Twins Discordant for Bipolar Disorder orSchizophreniaRachael J. Bloom1, Anna K Kähler1,2,3, Ann L. Collins1, Guanhua Chen4, Tyrone D.Cannon5, Christina Hultman2,†, and Patrick F. Sullivan1,2,6,†1Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA2Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden3Department of Psychiatry, Oslo University Hospital - Ulleval, Oslo, Norway4Department of Biostatistics University of North Carolina at Chapel Hill, Chapel Hill, NC, USA5Departments of Psychology and Psychiatry, Yale University, New Haven, CT, USA6Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAAbstractCopy number variation plays a clear role in the etiology of many psychiatric disorders, particularlyschizophrenia. We performed array-CGH to look for copy number variants between five pairs ofmonozygotic twins discordant for bipolar disorder or schizophrenia. Our study found nodifferences in copy number variants between the sets of twins. Although alluring, realisticaccounting for heterogeneity and chimerism highlight the technological limitations in studyingmonozygotic twins discordant for psychiatric disorders.Keywordsschizophrenia; bipolar disorder; monozygotic twin; discordant; genetics; copy number variation;structural variation© 2012 Elsevier B.V. All rights reserved.Correspond with Dr Sullivan: Department of Genetics, CB#7264, 5097 Genomic Medicine, University of North Carolina, Chapel Hill,NC, 27599-7264, USA. Voice: +919-966-3358, FAX: +919-966-3630, pfsulliv@med.unc.edu.†equivalent contributionsPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.AUTHOR CONTRIBUTIONSRJB performed the experiments and analysis and wrote paper. AKK and ALC helped design the experiment and interpret the results.GC conducted statistical analyses. TDC and CH designed and implemented the parent study. PFS designed the study and obtainedfunding. All authors reviewed and approved the final manuscript.CONFLICTS OF INTERESTDr Sullivan was on the SAB of Expression Analysis, Durham, NC. The other authors report no conflicts.NIH Public AccessAuthor ManuscriptSchizophr Res. Author manuscript; available in PMC 2014 May 01.Published in final edited form as:Schizophr Res. 2013 May ; 146(0): 289–290. doi:10.1016/j.schres.2013.02.021.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript1. IntroductionSchizophrenia and bipolar disorder have a concordance rate of about 50% in monozygotictwins, suggesting that both genetic and environmental factors contribute to the etiology ofthese psychiatric disorders.(Smoller and Finn, 2003; Sullivan et al., 2003) Copy numbervariants (CNVs) are an important class of genetic variation, and play a clear role in theetiology of many psychiatric disorders, particularly schizophrenia.(Sullivan et al., 2012) Inthis study, we analyzed CNV differences between monozygotic twins rigorously discordantfor a psychotic disorder to attempt to identify CNVs that arose mitotically after zygoteseparation that might be responsible for phenotypic differences.2. MethodsFull methods are in the Supplementary Materials. All protocols were reviewed and approvedby Ethical Committees in Sweden and the US, and all subjects provided written informedconsent. Briefly, using the population-based Swedish Twin Register(Lichtenstein et al.,2006), we ascertained and directly evaluated five pairs of monozygotic twins where onetwin met DSM-IV criteria for schizophrenia, schizoaffective disorder, or bipolar disorderand the cotwin had never met criteria for any psychotic disorder (Table S1). We furtherrequired that ≥10 years had passed since disease onset in the affected twin in order tominimize the effect of variable age of onset. Monozygosity was confirmed by genotyping apanel of 47 SNPs.(Hannelius et al., 2007) We analyzed DNA samples obtained from wholeblood using Roche Nimblegen 2.1M probe comparative genomic hybridization (CGH)arrays, with a median probe spacing of 1,169 bp, in theory sufficient for genome-widedetection of CNVs to ~20 Kb resolution. To enhance specificity, we used the dual-colorarray to hybridize the affected twin against the unaffected twin. Final CNV call sets weregenerated using NimbleScan (v2.6) with default settings. We removed segments with log2ratios from -0.26 to 0.22 because, under idealized assumptions, these limits are needed todetect one copy gains or one copy losses present in at least one-third of the cells in theaffected twin (Table S2). All predicted calls were annotated for intersections with genomegaps (telomeres and centromeres with low probe coverage can confound CNV callingalgorithms), segmental duplications, genes, and CNVs implicated in psychiatric disorders.(Sullivan et al., 2012)3. ResultsThere was a clear effect of array quality on the number of calls that were predicted. For ourchosen log2 ratio thresholds (Table S3). Using these log2 ratio thresholds, each pair had anaverage of 36 calls, with the number of calls ranging from 2 (Pairs 1 and 5) to 74 (Pair 3).We found no calls spanning gap regions. A few predicted calls overlapped with large (>1Mb) CNVs previously implicated in schizophrenia, but all of the predicted CNVs were small(<50 Kb) and overlapped very small proportions of these large CNVs (Table S4). Of the 186predicted calls, 67 contained genes. We visually reviewed all predicted calls but none hadclear breakpoints indicative of a true CNV but rather represented genomic “waviness”consistent with normal variation and noise in aCGH. One pair appeared to have aduplication of the entire transcript of SV2A (synaptic vesicle glycoprotein 2). We attemptedto verify this putative CNV using qPCR, but both twins appeared to be copy number two,suggesting a false positive aCGH CNV call (Figure S1).4. DiscussionMonozygotic twins discordant for disease have been the focus of many genetic andepigenetic studies.(Baranzini et al., 2010; Bruder et al., 2008; Maiti et al., 2011; Ono et al.,Bloom et al. Page 2Schizophr Res. Author manuscript; available in PMC 2014 May 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2010) As aCGH data have high-false positive and false-negative rates due to data quality,analysis methods, and other factors, (Scherer et al., 2007) we attempted to minimize theimpact of these factors with a rigorous set of manual and automated filters. Prior studies thatreported de novo CNVs discordant between MZ twins were discovered via comparison toother family members(Maiti et al., 2011) yet, to our knowledge, no prior study hashybridized MZ twins to each other. Other studies comparing genetic and epigeneticdifferences between MZ twins discordant for disease found no evidence of differences thatcould explain disease discordance.(Baranzini et al., 2010) Moreover, somatic mutations canbe mosaic within an individual, and an etiologically important CNV present in brain mightnot be found in peripheral blood. (Bruder et al., 2008)In choosing to hybridize the affected to the unaffected twin, we made several explicitassumptions. First, we attempted to maximize our chance of detection by directly searchingfor a CNV difference arising mitotically. Here, phenotypic discordance between MZ twins iscaused by a CNV present in the affected twin under a direct and parsimonious geneticmodel. It is possible that the true model is more complex. Given our hybridization choice,we would have missed detecting a germline CNV present in both the affected and unaffectedco-twin. This could have been important if, for example, a CNV is predisposing but requiresan environmental exposure to trigger the development of a psychotic disorder. Second, wehad DNA from a single tissue (peripheral blood). While we attempted to allow formosaicism, a causal CNV arising mitotically during development might exist only in a partof the brain and be absent in peripheral blood. Somatic mutations in the brains of individualswith hemimegalencephaly but not in peripheral blood have been described. (Poduri et al.,2012)In summary, we were unable to identify mitotic CNVs in peripheral blood that are candidateloci for phenotypic discordance between MZ twins rigorously discordant for psychoticdisorders. This work should be considered in light of two limitations. First, although weused a high-resolution aCGH platform, we still might have missed etiologically importantCNVs that were present in other tissues but not peripheral blood, not detectible by thetechnology we used, or which present in a small fraction of cells. Second, our sample sizewas small, and we cannot exclude the possibility of the existence of etiologically importantmitotic CNVs in schizophrenia. However, it is unlikely that large CNVs (>500Kb) werepresent in a large fraction of the somatic cells we studied, and we note that the offspring ofMZ twins discordant for schizophrenia appear to have similar risks for schizophrenia anobservation probably consistent with our negative results (Kringlen and Cramer, 1989).MZ twins rigorously discordant for psychosis represent a unique and compelling opportunityto search for causal genetic differences, but more conclusive study will require improvedtechnological resolution and direct access to alternative tissues. Under some genetic models,we note that this can be a challenging task for current genomic technologies (e.g.,identifying a single causal base pair change in a six billion base pair diploid genome presentin a localized portion of the brain).Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsROLE OF THE FUNDING SOURCEFunding for this project was from a NARSAD Distinguished Investigator award (Sullivan) the NIMH (MH052857,Cannon). The funders had no role in the design, execution, analysis, and manuscript preparation.Bloom et al. Page 3Schizophr Res. Author manuscript; available in PMC 2014 May 01.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptACKNOWLEDGEMENTS & ROLE OF THE FUNDING SOURCEFunding for this project was from a NARSAD Distinguished Investigator award (Sullivan) the NIMH (MH052857,Cannon). The funders had no role in the design, execution, analysis, and manuscript preparation.ReferencesBaranzini SE, Mudge J, van Velkinburgh JC, Khankhanian P, Khrebtukova I, Miller NA, Zhang L,Farmer AD, Bell CJ, Kim RW, May GD, Woodward JE, Caillier SJ, McElroy JP, Gomez R, PandoMJ, Clendenen LE, Ganusova EE, Schilkey FD, Ramaraj T, Khan OA, Huntley JJ, Luo S, KwokPY, Wu TD, Schroth GP, Oksenberg JR, Hauser SL, Kingsmore SF. Genome, epigenome and RNAsequences of monozygotic twins discordant for multiple sclerosis. Nature. 2010; 464(7293):1351–1356. [PubMed: 20428171]Bruder CE, Piotrowski A, Gijsbers AA, Andersson R, Erickson S, Diaz de Stahl T, Menzel U,Sandgren J, von Tell D, Poplawski A, Crowley M, Crasto C, Partridge EC, Tiwari H, Allison DB,Komorowski J, van Ommen GJ, Boomsma DI, Pedersen NL, den Dunnen JT, Wirdefeldt K,Dumanski JP. Phenotypically concordant and discordant monozygotic twins display different DNAcopy-numbervariation profiles. Am J Hum Genet. 2008; 82(3):763–771. [PubMed: 18304490]Hannelius U, Gherman L, Makela VV, Lindstedt A, Zucchelli M, Lagerberg C, Tybring G, Kere J,Lindgren CM. Large-scale zygosity testing using single nucleotide polymorphisms. Twin Res HumGenet. 2007; 10(4):604–625. [PubMed: 17708702]Kringlen E, Cramer G. Offspring of monozygotic twins discordant for schizophrenia. Arch GenPsychiatry. 1989; 46(10):873–877. [PubMed: 2802926]Lichtenstein P, Sullivan PF, Cnattingius S, Gatz M, Johansson S, Carlstrom E, Bjork C, SvartengrenM, Wolk A, Klareskog L, de Faire U, Schalling M, Palmgren J, Pedersen NL. The Swedish TwinRegistry in the third millennium: an update. Twin Res Hum Genet. 2006; 9(6):875–882. [PubMed:17254424]Maiti S, Kumar KH, Castellani CA, O'Reilly R, Singh SM. Ontogenetic de novo copy numbervariations (CNVs) as a source of genetic individuality: studies on two families with MZD twins forschizophrenia. PLoS One. 2011; 6(3):e17125. [PubMed: 21399695]Ono S, Imamura A, Tasaki S, Kurotaki N, Ozawa H, Yoshiura K, Okazaki Y. 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Comprehensive analysis of copy number variation in monozygotic twins discordant for bipolar disorder or schizophrenia

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Comprehensive analysis of copy number variation in monozygotic twins discordant for bipolar disorder or schizophrenia

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