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Aberrant DNA Methylation of Blood in Schizophrenia by Adjusting for Estimated Cellular Proportions

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Abstract

DNA methylation, which is the transference of a methyl group to the 5′-carbon position of the cytosine in a CpG dinucleotide, is one of the major mechanisms of epigenetic modifications. A number of studies have demonstrated altered DNA methylation of peripheral blood cells in schizophrenia (SCZ) in previous studies. However, most of these studies have been limited to the analysis of the CpG sites in CpG islands in gene promoter regions, and cell-type proportions of peripheral leukocytes, which may be one of the potential confounding factors for DNA methylation, have not been adjusted in these studies. In this study, we performed a genome-wide DNA methylation profiling of the peripheral leukocytes from patients with SCZ and from non-psychiatric controls (N = 105; 63 SCZ and 42 control subjects) using a quantitative high-resolution DNA methylation microarray which covered across the whole gene region (485,764 CpG dinucleotides). In the DNA methylation data analysis, we first estimated the cell-type proportions of each sample with a published algorithm. Next, we performed a surrogate variable analysis to identify potential confounding factors in our microarray data. Finally, we conducted a multiple linear regression analysis in consideration of these factors, including estimated cell-type proportions, and identified aberrant DNA methylation in SCZ at 2,552 CpG loci at a 5 % false discovery rate correction. Our results suggest that altered DNA methylation may be involved in the pathophysiology of SCZ, and cell heterogeneity adjustments may be necessary for DNA methylation analysis.

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References

  • Aberg, K. A., McClay, J. L., Nerella, S., Clark, S., Kumar, G., Chen, W., et al. (2014). Methylome-wide association study of schizophrenia: Identifying blood biomarker signatures of environmental insults. JAMA Psychiatry, 71, 255–264.

    Article  CAS  PubMed  Google Scholar 

  • Adalsteinsson, B. T., Gudnason, H., Aspelund, T., Harris, T. B., Launer, L. J., et al. (2012). Heterogeneity in white blood cells has potential to confound DNA methylation measurements. PLoS ONE, 7, e46705.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Aryee, M. J., Jaffe, A. E., Corrada-Bravo, J., Ladd-Acosta, C., Feinberg, A. P., et al. (2014). Minfi: A flexible and comprehensive Bioconductor package for the analysis of infinium DNA methylation microarrays. Bioinformatics, 30, 1363–1369.

    Article  CAS  PubMed  Google Scholar 

  • Bhutani, N., Burns, D. M., & Blau, H. M. (2011). DNA demethylation dynamics. Cell, 146, 866–872.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bibikova, M., Barnes, B., Tsan, C., Ho, V., Klotzle, B., Le, J. M., et al. (2011). High density DNA methylation array with single CpG site resolution. Genomics, 98, 288–295.

    Article  CAS  PubMed  Google Scholar 

  • Bock, C. (2012). Analysing and interpreting DNA methylation data. Nature Reviews Genetics, 13, 705–719.

    Article  CAS  PubMed  Google Scholar 

  • Bönsch, D., Wunschel, M., Lenz, B., Janssen, G., Weisbrod, M., & Sauer, H. (2012). Methylation matters? Decreased methylation status of genomic DNA in the blood of schizophrenic twins. Psychiatry Research, 198, 533–537.

    Article  PubMed  Google Scholar 

  • Carrard, A., Salzmann, A., Malafosse, A., & Karege, F. (2011). Increased DNA methylation status of the serotonin receptor 5HTR1A gene promoter in schizophrenia and bipolar disorder. Journal of Affective Disorders, 132, 450–453.

    Article  CAS  PubMed  Google Scholar 

  • Chen, Y., Lemire, M., Choufani, S., Butcher, D. C., Grafodatskaya, D., et al. (2013). Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray. Epigenetics, 8(2), 203–209.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen, Y., Zhang, J., Zhang, L., Shen, Y., & Xu, Q. (2012). Effects of MAOA promoter methylation on susceptibility to paranoid schizophrenia. Human Genetics, 131, 1081–1087.

    Article  CAS  PubMed  Google Scholar 

  • Cheng, J., Wang, Y., Zhou, K., Wang, L., Li, J., Zhuang, Q., et al. (2014). Male-specific association between dopamine receptor D4 gene methylation and schizophrenia. PLoS ONE, 9, e89128.

    Article  PubMed Central  PubMed  Google Scholar 

  • da Huang, W., Sherman, B. T., & Lempicki, R. A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols, 4, 44–57.

    Article  CAS  Google Scholar 

  • Dedeurwaerder, S., Defrance, M., Calonne, E., Denis, H., Sotiriou, C., & Fuks, F. (2011). Evaluation of the Infinium Methylation 450 K technology. Epigenomics, 3, 771–784.

    Article  CAS  PubMed  Google Scholar 

  • Dempster, E. L., Pidsley, R., Schalkwyk, L. C., Owens, S., Georgiades, A., Kane, F., et al. (2011). Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder. Human Molecular Genetics, 20, 4786–4796.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dong, E., Nelson, M., Grayson, D. R., Costa, E., & Guidotti, A. (2008). Clozapine and sulpiride but not haloperidol or olanzapine activate brain DNA demethylation. Proceedings of the National Academy of Sciences usa, 105, 13614–13619.

    Article  CAS  Google Scholar 

  • Gallia, G. L., Johnson, E. M., & Khalili, K. (2000). Puralpha: A multifunctional single-stranded DNA- and RNA-binding protein. Nucleic Acids Research, 28, 3197–3205.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gejman, P. V., Sanders, A. R., & Kendler, K. S. (2011). Genetics of schizophrenia: New findings and challenges. Annual Review of Genomics and Human Genetics, 12, 121–144.

    Article  CAS  PubMed  Google Scholar 

  • Guintivano, J., Aryee, M. J., & Kaminsky, Z. A. (2013). A cell epigenotype specific model for the correction of brain cellular heterogeneity bias and its application to age, brain region and major depression. Epigenetics, 8(3), 290–302.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Harvey, R. J., Carta, E., Pearce, B. R., Chung, S. K., Supplisson, S., Rees, M. I., et al. (2008). A critical role for glycine transporters in hyperexcitability disorders. Frontiers in Molecular Neuroscience, 1, 1–6.

    Article  PubMed Central  PubMed  Google Scholar 

  • Houseman, E. A., Accomando, W. P., Koestler, D. C., Christensen, B. C., Marsit, C. J., et al. (2012). DNA methylation array as surrogate measures of cell mixture distribution. BMC Bioinformatics, 13, 1471–2105.

    Article  Google Scholar 

  • Houseman, E. A., Molitor, J., & Marsit, C. J. (2014). Reference-free cell mixture adjustments in analysis of DNA methylation data. Bioinformatics, 30(10), 1431–1439.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Illingworth, R. S., Gruenewald-Schneider, U., Webb, S., Kerr, A. R. W., James, K. D., Turner, D. J., et al. (2010). Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genetics, 6, e1001134.

    Article  PubMed Central  PubMed  Google Scholar 

  • Irizarry, R. A., Ladd-Acosta, C., Wen, B., Wu, Z., Montano, C., Onyango, P., et al. (2009). The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nature Genetics, 41(2), 178–186.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jablensky, A., Sartorius, N., Ernberg, G., Anker, M., Korten, A., Cooper, J. E., et al. (1992). Schizophrenia: manifestations, incidence and course in different cultures. a world health organization ten-country study. Psychological Medicine. Monograph Supplement, 20, 1–97.

    Article  CAS  PubMed  Google Scholar 

  • Khojasteh-Fard, M., Tabrizi, M., & Amoli, M. M. (2011). Is DNA methylation responsible for immune system dysfunction in schizophrenia? Medical Hypotheses, 77, 573–579.

    Article  CAS  PubMed  Google Scholar 

  • Kim, J. G., Armstrong, R. C., Agoston, D. V., Wiese, A., Nagle, C., Hudson, J., et al. (1997). Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system. Journal of Neuroscience Research, 50(2), 272–290.

    Article  CAS  PubMed  Google Scholar 

  • Kinoshita, M., Numata, S., Tajima, A., Shimodera, S., Ono, S., Imamura, A., et al. (2013). DNA methylation signatures of peripheral leukocytes in schizophrenia. NeuroMolecular Medicine, 15, 95–101.

    Article  CAS  PubMed  Google Scholar 

  • Kordi-Tamandani, D. M., Sahranavard, R., & Torkamanzehi, A. (2012). DNA methylation and expression profiles of the brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes in patients with schizophrenia. Molecular Biology Reports, 39, 10889–10893.

    Article  CAS  PubMed  Google Scholar 

  • Kordi-Tamandani, D. M., Sahranavard, R., & Torkamanzehi, A. (2013). Evaluation of hypermethylation and expression pattern of GMR2, GMR5, GMR8, and GRIA3 in patients with schizophrenia. Gene, 515, 163–166.

    Article  CAS  PubMed  Google Scholar 

  • Lam, L. L., Emberly, E., Fraser, H. B., Neumann, S. M., Chen, E., et al. (2012). Factors underlying variable DNA methylation in a human community cohort. Proceedings of the National Academy of Sciences USA, 109, 17253–17260.

    Article  CAS  Google Scholar 

  • Lambert, S. R., Witt, H., Hovestadt, V., Zucknick, M., Kool, M., Pearson, D. M., et al. (2013). Differential expression and methylation of brain developmental genes define location-specific subsets of pilocytic astrocytoma. Acta Neuropathologica, 126(2), 291–301.

  • Langevin, S. M., Houseman, E. A., Accomando, W. P., Koestler, D. C., Christensen, B. C., Nelson, H. H., et al. (2014). Leukocyte-adjusted epigenome-wide association studies of blood from solid tumor patients. Epigenetics, 9(6), 884–895.

  • Lee, Y., Mattai, A., Long, R., Rapoport, J. L., Gogtay, N., & Addington, A. M. (2012). Microduplications disrupting the MYT1L gene (2p25.3) are associated with schizophrenia. Psychiatric Genetics, 22(4), 206–209.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Leek, J. T., & Storey, J. D. (2007). Capturing heterogeneity in gene expression studies by surrogate variable analysis. PLoS Genetics, 3, 1724–1735.

    Article  CAS  PubMed  Google Scholar 

  • Li, W., Wang, X., Zhao, J., Lin, J., Song, X. Q., Yang, Y., et al. (2012). Association study of myelin transcription factor 1-like polymorphisms with schizophrenia in Han Chinese population. Genes, Brain and Behavior, 11(1), 87–93.

    Article  Google Scholar 

  • Liu, Y., Aryee, M. J., Padyukov, L., Fallin, M. D., Hesselberg, E., et al. (2013). Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis. Nature Biotechnology, 31, 142–147.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Maunakea, A. K., Nagarajan, R. P., Bilenky, M., Ballinger, T. J., D’souza, C., Fouse, S. D., et al. (2010). Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature, 466, 253–257.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Melas, P. A., Rogdaki, M., Ösby, U., Schalling, M., Lavebratt, C., & Ekström, T. J. (2012). Epigenetic aberrations in leukocytes of patients with schizophrenia: association of global DNA methylation with antipsychotic drug treatment and disease onset. The FASEB Journal, 26, 2712–2718.

    Article  CAS  Google Scholar 

  • Murata, Y., Nishioka, M., Bundo, M., Sunaga, F., Kasai, K., & Iwamoto, K. (2014). Comprehensive DNA methylation analysis of human neuroblastoma cells treated with blonanserin. Neuroscience Letters, 20(563), 123–128.

    Article  Google Scholar 

  • Nakatani, N., Hattori, E., Ohnishi, T., Dean, B., Iwayama, Y., Matsumoto, I., et al. (2006). Genome-wide expression analysis detects eight genes with robust alterations specific to bipolar I disorder: relevance to neuronal network perturbation. Human Molecular Genetics, 15, 1949–1962.

    Article  CAS  PubMed  Google Scholar 

  • Nielsen, J. A., Berndt, J. A., Hudson, L. D., & Armstrong, R. C. (2004). Myelin transcription factor 1 (Myt1) modulates the proliferation and differentiation of oligodendrocyte lineage cells. Molecular and Cellular Neuroscience, 25, 111–123.

    Article  CAS  PubMed  Google Scholar 

  • Nishioka, M., Bundo, M., Koike, S., Takizawa, R., Kakiuchi, C., Araki, T., et al. (2013). Comprehensive DNA methylation analysis of peripheral blood cells derived from patients with first-episode schizophrenia. Journal of Human Genetics, 58, 91–97.

    Article  CAS  PubMed  Google Scholar 

  • Numata, S., Ye, T., Hyde, T. M., Guitart-Navarro, X., Tao, R., Wininger, M., et al. (2012). DNA methylation signatures in development and aging of the human prefrontal cortex. American Journal of Human Genetics, 90, 260–272.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Petronis, A., Gottesman, I. I., Crow, T. J., DeLisi, L. E., Klar, A. J., Macciardi, F., et al. (2000). Psychiatric epigenetics: A new focus for the new century. Molecular Psychiatry, 5, 342–346.

    Article  CAS  PubMed  Google Scholar 

  • Rao, X., Evans, J., Chae, H., Pilrose, J., Kim, S., Yan, P., et al. (2013). CpG island shore methylation regulates caveolin-1 expression in breast cancer. Oncogene, 32(38), 4519–4528.

  • Reinius, L. E., Acevedo, N., Joerink, M., Pershagen, G., Dahlen, S. E., et al. (2012). Differential DNA methylation in purified human blood cells: Implications for cell lineage and studies on disease susceptibility. PLoS ONE, 7, e41361.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Roth, T. L., & Sweatt, J. D. (2011). Annual research review: Epigenetic mechanisms and environmental shaping of the brain during sensitive periods of development. Journal of Child Psychology and Psychiatry, 52, 398–408.

    Article  PubMed Central  PubMed  Google Scholar 

  • Rutter, M., Moffitt, T. E., & Caspi, A. (2006). Gene-environment interplay and psychopathology: Multiple varieties but real effects. Journal of Child Psychology and Psychiatry, 47, 226–261.

    Article  PubMed  Google Scholar 

  • Sandoval, J., Heyn, H. A., Moran, S., Serra-Musach, J., Pujana, M. A., Bibikova, M., et al. (2011). Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome. Epigenetics, 6, 692–702.

    Article  CAS  PubMed  Google Scholar 

  • Siegmund, K. D., Connor, C. M., Campan, M., Long, T. I., Weisenberger, D. J., Biniszkiewicz, D., et al. (2007). DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons. PLoS ONE, 2, e895.

    Article  PubMed Central  PubMed  Google Scholar 

  • Teschendorff, A. E., Zhuang, J., & Widschwendter, M. (2011). Independent surrogate variable analysis to deconvolve confounding factors in large-scale microarray profiling studies. Bioinformatics, 27(11), 1496–1505.

    Article  CAS  PubMed  Google Scholar 

  • Tolosa, A., Sanjuán, J., Dangnall, A. M., Moltó, M. D., Herrero, N., & de Frutos, R. (2010). FOXP2 gene and language impairment in schizophrenia: Association and epigenetic studies. BMC Medical Genetics, 11, 114.

    Article  PubMed Central  PubMed  Google Scholar 

  • Tsai, G. E., & Lin, P. Y. (2010). Strategies to enhance N-methyl-D-aspartate receptor-mediated neurotransmission in schizophrenia, a critical review and meta-analysis. Current Pharmaceutical Design, 16(5), 522–537.

    Article  CAS  PubMed  Google Scholar 

  • Van Den Bossche, M. J., Strazisar, M., Cammaerts, S., Liekens, A. M., Vandeweyer, G., Depreeuw, V., et al. (2013). Identification of rare copy number variants in high burden schizophrenia families. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 162B(3), 273–282.

    Article  Google Scholar 

  • Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Südhof, T. C., & Wernig, M. (2010). Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 463(7284), 1035–1041.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vrijenhoek, T., Buizer-Voskamp, J. E., van der Stelt, I., Strengman, E., Genetic Risk and Outcome in Psychosis (GROUP) Consortium, Sabatti, C., et al. (2008). Recurrent CNVs disrupt three candidate genes in schizophrenia patients. The American Journal of Human Genetics, 83(4), 504–510.

    Article  CAS  Google Scholar 

  • Xu, H., Wang, B., Su, D., Yu, Q., Li, Q., Kou, C., et al. (2012). The DNA methylation profile of PLA2G4C gene promoter in schizophrenia. Psychiatry Research, 200, 1079–1081.

    Article  CAS  PubMed  Google Scholar 

  • Zouridis, H., Deng, N., Ivanova, T., Zhu, Y., Wong, B., Huang, D., et al. (2012). Methylation subtypes and large-scale epigenetic alterations in gastric cancer. Science Translational Medicine, 4, 156.

    Article  Google Scholar 

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Acknowledgments

The authors thank Dr. Jörg Tost for his valuable comments and suggestions on SNP-associated probes in the Illumina HumanMethylation450 platform. The authors would also like to thank Mrs. Akemi Okada and Mrs. Kumiko Kikuchi for their technical assistance. The authors appreciate all the volunteers who understood our study purpose and participated in this study, and the physicians who helped us to collect clinical data and blood samples at the mental hospitals. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant Number 24791216), SENSHIN Medical Research Foundation, and the Research Group for Schizophrenia.

Conflict of interest

The all authors report no biomedical financial interests or potential conflicts of interest.

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Authors and Affiliations

  1. Department of Psychiatry, Course of Integrated Brain Sciences, Medical Informatics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-8-15, Kuramoto-cho, Tokushima, 770-8503, Japan

    Makoto Kinoshita, Shusuke Numata & Tetsuro Ohmori

  2. Department of Human Genetics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-8-15, Kuramoto-cho, Tokushima, 770-8503, Japan

    Atsushi Tajima & Issei Imoto

  3. Department of Psychiatry, Osaka University Graduate School of Medicine, D3, 2-2, Yamadaoka, Suita, Osaka, 565-0871, Japan

    Kazutaka Ohi & Ryota Hashimoto

  4. Department of Neuropsychiatry, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi, 783-8505, Japan

    Shinji Shimodera

  5. Molecular Research Center for Children’s Mental Development, United Graduate School of Child Development, Osaka University, D3, 2-2, Yamadaoka, Suita, Osaka, 565-0871, Japan

    Ryota Hashimoto & Masatoshi Takeda

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  1. Makoto Kinoshita
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  2. Shusuke Numata
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  3. Atsushi Tajima
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  4. Kazutaka Ohi
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  9. Tetsuro Ohmori
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Correspondence to Shusuke Numata.

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12017_2014_8319_MOESM1_ESM.tif

DNA methylation levels of three CpG sites in the AR gene. The AR gene is a X-linked gene. Three CpG sites in CpG islands in the AR gene showed higher DNA methylation in female. (TIFF 391 kb)

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Kinoshita, M., Numata, S., Tajima, A. et al. Aberrant DNA Methylation of Blood in Schizophrenia by Adjusting for Estimated Cellular Proportions. Neuromol Med 16, 697–703 (2014). https://doi.org/10.1007/s12017-014-8319-5

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