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Methylation Marks Hypoxia in Schizophrenia

January 30, 2014. The first genomewide survey of methylation patterns in schizophrenia turns up 25 suspect regions, some of which include genes linked to hypoxia. Published online January 8 in JAMA Psychiatry, the study examines blood samples from 759 people with schizophrenia and 738 controls to find differences in the methyl groups that, when attached to a segment of DNA, suppress expression of nearby genes. Led by Edwin van den Oord at Virginia Commonwealth University in Richmond, the results suggest that methylation patterns found in blood could record past environmental insults and may serve as useful biomarkers for schizophrenia.

Some methylation patterns found in blood cells may mirror those in the brain, according to a second, smaller study published in Translational Psychiatry on January 7. A team led by Joanne Voisey at Queensland University of Technology, Brisbane, Australia, found that 99 genes that were differently methylated in brain samples from people with schizophrenia compared to controls were on the same list of genes previously singled out as differently methylated in blood samples.

Together, the results point to methylation patterns as a rich source for variation that may hold clues to how genes and environment combine to boost risk for schizophrenia. Researchers have seized upon epigenetics, either in the form of methylation or histone modification, because it offers a way for experience to alter what is produced by the genome. The cells in which the methylation patterns are studied, however, may matter. If patterns found in blood match those found in the brain, they might provide some causal insights into the disease process. If they are specific to blood, however, they may still be a useful biomarker from an easy to obtain source.

Previous studies have found methylation differences in schizophrenia in a limited selection of genes (see SRF related news report), but, as reported from a conference last year (see SRF related conference report), the new study is the first to look at all methylation across the entire genome, termed the “methylome.” Using a design similar to genomewide association studies (GWAS) (see SRF genetics primer), the researchers systematically compared methylation sites between cases and controls to see if any were differently methylated. Unlike initial GWAS efforts, the resulting methylome-wide association study (MWAS) identified some definitive hits that were also replicated in a second group.

Hints of hypoxia
First author Karolina Aberg and colleagues began by extracting the methylated portions of the genome from their samples, then sequencing those parts. They checked the methylation status of each of 26,752,702 methylation sites, also known as “CpG” sites, for the sequence of cytosine and guanine that permits methylation. Because the methylation status of a single site can be highly correlated with adjacent methylation sites, the researchers collapsed adjacent sites into blocks, giving a total of 4,344,016 blocks, which they tested for association with schizophrenia.

In all, 139 of these were significantly associated with schizophrenia, with 112 of the sites residing within genes. Some of these genes, such as SMAD3 and ARNT, are affected by hypoxia, with their transcription varying with oxygen levels. Network analyses of all MWAS-significant genes found that they were enriched in hypoxia-related gene sets, as well as immune system genes, based on interactions between their protein products, the pathways to which they belonged, or whether they were targets of the same microRNAs.

Twenty-five of the 139 survived a conservative correction for multiple comparisons, and the top hit—FAM63B—was replicated in an independent cohort. FAM63B itself is regulated by different microRNAs, which the researchers noted have links to neuron differentiation and dopamine expression. RELN, a familiar candidate gene found to be overly methylated in schizophrenia (Grayson et al., 2005), also turned up as a significant hit in the MWAS, and this remained significant in their replication sample. Except for RELN, the sites showed decreased methylation in schizophrenia relative to controls.

Deep past?
Smoking and other lifestyle differences did not explain the results, leaving the researchers to speculate that methylation status over hypoxia-related genes reflected past environmental mishaps. These may contribute to schizophrenia risk, which is boosted by obstetric complications (e.g., Clarke et al., 2011; see SRF related news report). The methylation patterns over these hypoxia-related genes would not necessarily be the risk factor incarnate; rather, they would compose a signature of past, pathogenic events. Even if such a signature remained only in blood cells, the researchers argue it could still provide a useful biomarker for identifying subtypes of schizophrenia.

Alternatively, blood may be a good proxy for brain, as suggested by the RELN findings, which mimicked those in earlier studies of postmortem brain. This view was supported by Voisey’s study of a less extensive set of methylation sites in postmortem brain samples from 24 people with schizophrenia and 24 controls. When first author L. Wockner and colleagues scanned 485,000 CpG sites for methylation, they found differences in 2,929 genes between schizophrenia and controls. Some of these, such as NOS1, SOX10, and DTNBP1, have previous ties to schizophrenia, and 99 were in the list of 589 genes flagged by a previous methylation study in blood (Nishioka et al., 2012). These results suggest that, to some extent, methylation patterns in blood may mirror the situation in brain and may provide clues to causal factors in schizophrenia.—Michele Solis.

Aberg KA, McClay JL, Nerella S, Clark S, Kumar G, Chen W, Khachane AN, Xie L, Hudson A, Gao G, Harada A, Hultman CM, Sullivan PF, Magnusson PK, van den Oord EJ. Methylome-Wide Association Study of Schizophrenia: Identifying Blood Biomarker Signatures of Environmental Insults. JAMA Psychiatry. 2014 Jan 8. Abstract

Wockner LF, Noble EP, Lawford BR, Young RM, Morris CP, Whitehall VL, Voisey J. Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients. Transl Psychiatry. 2014 Jan 7. Abstract

Comments on Related News

Related News: Epigenetic Analysis Finds Widespread DNA Methylation Changes in Psychosis

Comment by:  Dennis Grayson
Submitted 26 March 2008
Posted 27 March 2008
  I recommend the Primary Papers

The paper by Mill et al. is one of the first comprehensive attempts to examine changes in methylation across the entire genome in patients with various diagnoses of mental illness. The study is well designed, extensive, and uses fairly new technology to examine changes in methylation profiles across the genome. In the frontal cortex, the authors provide evidence for psychosis-associated differences in DNA methylation in numerous loci, including those involved in glutamatergic and GABAergic transmission, brain development, and other processes linked with disease etiology. Methylation in the frontal cortex of the BDNF gene is correlated with a non-synonymous SNP previously associated with major psychosis. These data provide further support for an epigenetic origin of major psychosis, as evidenced by DNA methylation-induced changes likely important to gene expression.

In many ways, this seems reminiscent of the trend in genetics several years ago when the inclination was to move from single gene loci association and linkage studies to genomewide scans. The only downside of the approach is that what one gains in information, one (at least initially) loses in biology. That is given the wealth of new findings uncovered; we now need to go back and examine these results in light of what we know regarding gene function in neurobiology and cognition. Of course, this is the trend, now that microarrays have increased our capacity to look at all things at the same time. The flipside is that it will take several large-scale studies of this sort to better understand which findings are replicable and which are not. That is, do the results of the Mill paper agree with data obtained and carried out by laboratories using the methyl DIP or MeCP2 ChIP assays coupled with microarrays. While these experiments ask different questions, the implication is that there may be some degree of overlap in comparing these different methodologies. While this may be premature, there is a sense that this information will be available shortly.

Finally, I would like to focus on recent findings regarding the methylation of the reelin promoter. These authors (Mill et al.) and Tochigi and colleagues (Tochigi et al., 2008) have found that the reelin promoter is not hypermethylated in patients with schizophrenia. In fact, Tochigi et al., 2008, found that the reelin promoter is not methylated at all using pyrosequencing. However, several groups (Grayson et al., 2005; Abdolmaleky et al., 2005; Tamura et al., 2007; Sato et al., 2006) have shown that the human reelin promoter is methylated in different circumstances. Interestingly, there is little consensus in the precise bases that are methylated in these latter studies. Our group (Grayson et al., 2005) performed bisulfite treatment of genomic DNA and sequencing of individual clones. Moreover, we analyzed two distinct patient populations. The clones were sequenced at a separate facility. What was intriguing was that the baseline methylation patterns in the two populations was different, and yet several sites stood out as being relevant in both. We mapped methylation to the somewhat rare CpNpG sites proximal to the promoter. Interestingly, these bases were located in a transcription factor-rich portion (Chen et al., 2007) of the promoter and in a region that shows 100 percent identity with the mouse promoter over a 45 bp stretch. We have also been able to show that changing one of these two bases to something other than cytosine reduces activity 50 percent in a transient transfection assay. So the question becomes, How do we reconcile these disparate findings regarding methylation? As suggested by Dr. McCaffrey, the answer may lie in regional differences that arise due to the nature of the material available for each study. We have found a degree of reproducibility by using human neuronal precursor (NT2) cells for many of our studies. At the same time, this cell line is somewhat artificial and cannot be used to reconcile differences found in human tissue. Perhaps it might be prudent to examine material taken by using laser capture microdissection to enrich in more homogenous populations of neurons/glia. In moving ahead, it might be best to now focus on the mechanism for these differences in methylation patterns and try to understand the biology associated with the new findings (Mill et al., 2008) as a starting point.


Abdolmaleky HM, Cheng KH, Russo A, Smith CL, Faraone SV, Wilcox M, Shafa R, Glatt SJ, Nguyen G, Ponte JF, Thiagalingam S, Tsuang MT. Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report. Am J Med Genet B Neuropsychiatr Genet. 2005 Apr 5;134(1):60-6. Abstract

Chen Y, Kundakovic M, Agis-Balboa RC, Pinna G, Grayson DR. Induction of the reelin promoter by retinoic acid is mediated by Sp1. J Neurochem. 2007 Oct 1;103(2):650-65. Abstract

Grayson DR, Jia X, Chen Y, Sharma RP, Mitchell CP, Guidotti A, Costa E. Reelin promoter hypermethylation in schizophrenia. Proc Natl Acad Sci U S A. 2005 Jun 28;102(26):9341-6. Abstract

Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, Jia P, Assadzadeh A, Flanagan J, Schumacher A, Wang SC, Petronis A. Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. Am J Hum Genet. 2008 Mar 1;82(3):696-711. Abstract

Sato N, Fukushima N, Chang R, Matsubayashi H, Goggins M. Differential and epigenetic gene expression profiling identifies frequent disruption of the RELN pathway in pancreatic cancers. Gastroenterology. 2006 Feb 1;130(2):548-65. Abstract

Tamura Y, Kunugi H, Ohashi J, Hohjoh H. Epigenetic aberration of the human REELIN gene in psychiatric disorders. Mol Psychiatry. 2007 Jun 1;12(6):519, 593-600. Abstract

Tochigi M, Iwamoto K, Bundo M, Komori A, Sasaki T, Kato N, Kato T. Methylation status of the reelin promoter region in the brain of schizophrenic patients. Biol Psychiatry. 2008 Mar 1;63(5):530-3. Abstract

View all comments by Dennis Grayson

Related News: Schizophrenia Genetics 2: The Rise of GWAS

Comment by:  Chris Carter
Submitted 7 April 2010
Posted 8 April 2010

I wonder whether the relative lack of success in schizophrenia GWAS may be because the origin of schizophrenia may lie not so much in the genetic make-up of people with schizophrenia themselves, but in their prenatal experience, and possibly with the genes of the mother rather than with those of the offspring. Famine, rubella, influenza, herpes (HSV1 and HSV2), and poliovirus infection as well as high fever during pregnancy have all been listed as risk factors for the offspring developing schizophrenia in later life, as have maternal preeclampsia and obstetric complications. (See page at Polygenic Pathways for the many references.)

Maternal resistance to these effects is likely to be gene-dependent. Is it worth considering GWAS in the mothers rather than in the offspring?

View all comments by Chris Carter

Related News: Searching for the Elusive Epigenetic Influence on Schizophrenia

Comment by:  Irving Gottesman, SRF Advisor
Submitted 12 May 2016
Posted 16 May 2016
  I recommend the Primary Papers

I, for one, want to express my gratitude to Michele Solis for the clarity she brings to finding the trees in the forests of dense journal reporting demanded by word-length handcuffs.

View all comments by Irving Gottesman