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Twins Tell Story of Epigenetic Alterations in Schizophrenia and Bipolar Disorder

6 October 2011. DNA methylation changes in monozygotic twins discordant for schizophrenia and bipolar disorder provide strong evidence for a role of epigenetic alterations in psychosis, according to a new study published online September 22 in Human Molecular Genetics. In the first large-scale, genomewide analysis of epigenetic changes in twin pairs discordant for psychosis, Jonathan Mill and Robin Murray, of King’s College London, and colleagues report altered DNA methylation patterns associated with both schizophrenia and bipolar disorder.

The high heritability of psychotic disorders is widely recognized, although poorly understood (Craddock et al., 2005). The vast majority of studies have examined the role of genes in schizophrenia and bipolar disorder, although recently epigenetic alterations—mitotically heritable changes that control gene activity without altering the DNA sequence—have garnered attention in these illnesses (Pidsley and Mill, 2011: see also SRF Current Hypothesis paper by D. Grayson). Epigenetic changes are principally brought about through DNA methylation at cytosine-guanine dinucleotide (CpG) sites and chromatin alterations (Henikoff and Matzke, 1997).

Mill's group has previously reported DNA methylation changes in schizophrenia and bipolar disorder (see SRF related news story) in a large number of unrelated individuals. The study of monozygotic twins discordant for psychosis is a particularly powerful approach, since epigenetic changes can be measured independent of variation in genetic code (Bell and Spector, 2011). This approach has been used successfully outside the field (Javierre et al., 2010); however, similar studies of psychosis have been limited to small sample sizes of only a few twin pairs (Petronis et al., 2003; Kuratomi et al., 2008).

Using peripheral blood samples from 22 twin pairs (11 pairs each discordant for schizophrenia and bipolar disorder, respectively), first authors Emma Dempster and Ruth Pidsley performed a genomewide analysis of DNA methylation at CpG sites using microarray. A number of disease-associated changes were found, and a pathway analysis of top loci revealed that epigenetic changes were prominent in a number of networks related to psychiatric illness and neurodevelopment. Additionally, methylation alterations were also found at loci not previously implicated in either illness. Some DNA methylation differences were present across all twin pairs, while others appeared to be more rare (appearing in only a single pair or two), suggesting that certain epigenetic alterations in psychotic illness may be more widespread than others.

Interestingly, a number of epigenetic differences were found across a combined psychosis group, while others were specific to schizophrenia or bipolar disorder. When all pairs were pooled together into a psychosis group, resulting in the largest sample size, ST6GALNAC1, a gene involved in protein glycosylation, emerged as the most differentially methylated region. Hypomethylation of this CpG site was confirmed using postmortem brain samples from the Stanley Foundation Neuropathology Consortium. Given that hypomethylation typically leads to gene overexpression, the results of the current study are consistent with a duplication in ST6GALNAC1 that was recently implicated in schizophrenia (Xu et al., 2008; see SRF related news story).

When the twin pairs were broken down into schizophrenia and bipolar disorder groups, differences emerged. The top hit in schizophrenia twin pairs was a CpG site located upstream of the gene encoding pseudouridylate synthase 3 (PUS3), which was hypermethylated in affected twins. In contrast, in bipolar disorder twin pairs, the largest difference was a hypomethylation within a G protein-coupled receptor gene, GPR24, which has previously been implicated as a susceptibility gene for both schizophrenia and bipolar disorder (Severinsen et al., 2006). Of note, opposite methylation patterns were also found between the two illnesses at the same sites, such as the hypermethylation of the promoter of zinc finger gene ZNF659 in schizophrenia twins but hypomethylation in bipolar twins.

This study further implicates epigenetic alterations in the etiology of psychotic illness. Given the success of the current methodology in detecting alterations in methylation patterns, the authors note that “future efforts should focus on collecting biological material from additional twin pairs for use in methylomic analyses.”—Allison Curley.

Dempster EL, Pidsley R, Schalkwyk LC, Owens S, Georgiades A, Kane F, Kalidindi S, Picchioni M, Kravariti E, Toulopoulou T, Murray RM, Mill J. Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder. Hum Mol Genet . 2011 Sep 22. Abstract

Comments on News and Primary Papers
Comment by:  Schahram Akbarian
Submitted 7 October 2011
Posted 7 October 2011

The genetic risk architecture is still very difficult to "capture" for a large majority of patients diagnosed with schizophrenia or related diseases. Therefore, studies like that of Dempster et al., who profiled DNA methylation (a type of "epigenetic" modification of cytosine that residues mostly at sites of CpG dinucleotides in the genome) in blood cells of monozygotic twins discordant for schizophrenia, provide an important additional layer of information. The idea is that the disease process in the affected twin leaves behind a molecular signature (in the study by Dempster et al., this would be a change in DNA methylation) that is not found in the healthy twin, with the implication that this signal is related to disease etiology or disease process and treatment, etc.

Dempster and colleagues screened approximately 20 twin pairs. I believe the Illumina bead system they used probes primarily annotated promoters; on a genomewide level, they found, overall, quite subtle changes. One of the more prominent findings is hypomethylation of one specific CpG dinucleotide associated with ST6GALNAC1, a gene regulating protein glycosylation, with additional changes in a dozen or so genes. Hypomethylation of ST6GALNAC1 (which the authors further verified in postmortem brains of subjects with schizophrenia) at sequences proximal to promoters is generally associated with increased expression, but it is not clear if this is the case in the twin blood or the postmortem brain. Interestingly, as the authors point out, the same gene (ST6GALNAC1) may harbor an excess copy in some subjects on the psychosis spectrum. This is a good example of the possibility that some of the genes that are potentially linked to psychosis because of DNA sequence and copy number changes may also show up in epigenetic studies such as that of Dempster and colleagues. Whether a genetic variation or mutation somewhere else in the genome "drives" the epigenetic changes at ST6GALNAC1 and other genes is, of course, hard to prove.

The most important challenge to the epigenetics and psychosis fields is, at least in my opinion, that of reproducibility and independent replication. In other words, will we be able to replicate, in independent studies, epigenetic alterations in blood or postmortem brain tissue, reported to be "highly significant" for cohorts comprising a few dozen or fewer cases? With fewer than five published studies (to the best of my knowledge) that measured DNA methylation on a genomewide scale in mood and psychosis spectrum disorders, it is still hard to predict whether DNA and histone modification mapping will provide valuable clues to the underlying neurobiology of disease. I take an optimistic view, and would like to predict that DNA methylation and histone modification mapping will provide a very important additional layer of information when paired with whole-genome sequencing and transcriptome (RNA expression) profiling.

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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.


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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

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