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Epigenetic Forces May Blaze Divergent Heritable Paths From Same DNA

27 January 2009. The mystery of why one twin develops schizophrenia and the other one does not, even when they share all of the same genes, has persisted despite the search for environmental explanations. A new study, reported online in Nature Genetics on January 18, offers clues. Conducted by Art Petronis of the University of Toronto and the Centre for Addiction and Mental Health in Toronto, it found vast differences in gene regulation across the genome, even in monozygotic twins. Most strikingly, Petronis and colleagues report that these epigenetic differences may arise from a second molecular mechanism of heritability other than variations in the DNA sequence.

Epigenetic processes control gene activity without changing the genetic code. While the DNA sequence sets a stable “stage,” epigenetic processes correspond to a “play” in progress, according to Petronis (Petronis, 2004).

Recent findings suggest that epigenetic mechanisms play key roles in neural development, activity, and survival (see review by Tsankova et al., 2007; also see SRF related news story). At least one hypothesis has cast them as causal agents in schizophrenia, based in part on observations that methionine, an amino acid that alters gene expression, triggers psychotic symptoms in patients with schizophrenia (see SRF Current Hypothesis by Grayson and colleagues).

One epigenetic mechanism involves attaching methyl groups to DNA (see SRF related news story; for another epigenetic process, see SRF related news story on histone methylation). A genomewide scan last year by Petronis's group found abnormal DNA methylation patterns in frontal cortex tissue obtained from subjects with schizophrenia or bipolar disorder (see SRF related news story).

Previously, the Petronis lab found DNA methylation differences between single-egg co-twins and suggested that these might explain why about half will show discordance for schizophrenia (Petronis et al., 2003). The new study takes a more comprehensive look at cells’ epigenetic state. It examined DNA methylation at specific loci across the entire genome in 114 monozygotic and 80 dizygotic twins.

Led by first author Zachary Kaminsky, also of the Centre for Addiction and Mental Health and the University of Toronto, the researchers examined cheek, gut, and white blood cells. The gut cells came from non-inflamed tissue collected during biopsies for a study of inflammatory bowel disease. In an interview with SRF, Irving Gottesman of the University of Minnesota in Minneapolis, one of the study’s collaborators, said, “Ideally, we would have had brain tissue,” but that can be hard to obtain.

Kaminsky and colleagues used microarrays to explore the unmethylated genome on CpG islands, cytosine-and-guanine packed areas where methylation can silence genes. In all three kinds of tissue, they found DNA methylation differences between monozygotic co-twins. The greatest dissimilarities occurred in cheek cells of identical co-twins who resulted from a later, rather than early, splitting of the embryo. The researchers suggest that phenotypic similarities between genetically identical co-twins may reflect epigenetic factors that they share at the time the blastocyst divides.

Despite these differences between monozygotic siblings, their gene expression patterns looked more alike than those of dizygotic co-twins, although this finding turned out to be due entirely to the early-splitting group of same-egg twins. Since DNA sequence differences between identical twins rarely occur, Kaminsky and associates think it unlikely that their widespread epigenetic dissimilarities could follow from the genetic code; rather, they must reflect inherited gene regulation profiles. This counters the long-held “truth” that DNA sequence variations represent the sole means of passing heritable information between generations.

Marshalling additional support for that conclusion, Kaminsky and associates noted that, unlike the DNA sequence in a given individual, methylation patterns varied by tissue type. Furthermore, inbred mice, who share nearly identical genes, showed similar methylation profiles to outbred mice.

Just as DNA fingerprinting has reopened criminal cases, the discovery of this other kind of heritability, if replicated, may signal the need to revisit twin studies. According to Gottesman, who conducted many of the seminal heritability studies in mental illness, “Now that we have noticed these phenomena under the umbrella of epigenetics, it may be that we’ve overlooked some of the most important factors in the etiology of schizophrenia and other major mental disorders that have an important genetic component, as inferred from twin and ordinary family studies.”—Victoria L. Wilcox.

Kaminsky ZA, Tang T, Wang S-C, Ptak C, Oh GHT, Wong AHC, Feldcamp LA, Virtanen C, Halfvarson J, Tysk C, McRae AF, Visscher PM, Montgomery GW, Gottesman II, Martin NG, Petronis A. DNA methylation profiles in monozygotic and dizygotic twins. Nat Genet. 2009 Jan 18. Abstract

Q&A With Art Petronis. Questions by Hakon Heimer and Victoria Wilcox.

Q: To start off, could you summarize the most important findings and your conclusions from them?
A: The most important finding is that, although we still don’t have the final answer, we cannot exclude a possibility that epigenetics may be a secondary molecular substrate of heritability. Most, if not all, human diseases, including all psychiatric diseases, exhibit evidence for inherited predisposition. The most elegant way to estimate heritability is by comparison of concordance of monozygotic (MZ) and dizygotic (DZ) twins. In schizophrenia, the numbers are as follows: MZ twins exhibit 50 percent concordance, while DZ twins exhibit 15 percent concordance for schizophrenia, and the heritability is 70 percent. The traditional understanding is that DNA sequence is the molecular basis of heritability.

In our recent twin study, we compared the degree of epigenetic differences between MZ twins to the degree of epigenetic differences between DZ twins. We detected that MZ twins, despite their numerous epigenetic differences, are still more similar to each other in comparison to DZ twins. There could be several explanations for this. The null hypothesis is that the larger epigenetic differences in DZ twins are secondary; i.e., they are induced by DNA sequence differences in DZ twins. The alternative hypothesis is that those large epigenetic differences that we see in DZ twins represent vestiges of the inherited epigenetic profiles. MZ twins originate from a single zygote and, therefore, their epigenetic origin is the same (or very similar). DZ twins originate from two separate zygotes, the epigenetic profiles of which can be very different. Before continuing, it is necessary to explain the concept of epigenetic variation of the zygotes. In our earlier studies, we investigated the epigenetics of the germline, more specifically, sperm, and detected significant epigenetic differences across sperm cells from the same individual. Even short (about 500 nucleotide) stretches of DNA exhibited enough epigenetic variation to make each germ cell unique from the epigenetic point of view. We don’t know if oocytes exhibit such a significant degree of epigenetic variation, but it is likely to be similar to what we detected in the sperm. If germ cells are different epigenetically, zygotes will be different as well.

We performed a series of additional experiments looking at the possible effects of DNA sequence on DNA methylation and did not detect much evidence that the larger epigenetic differences in DZ twins are secondary. These findings favor—and I want to emphasize that they do not prove, but only favor—the alternative hypothesis that some of the epigenetic peculiarities in the zygotes are not completely erased; they survive all those numerous mitotic divisions experienced by an organism during development. If some of the inherited epigenetic factors play a role in phenotypic outcomes, we have a case of epigenetic heritability.

The ramifications of this finding may be quite interesting. Traditional genetic studies in complex diseases thus far could explain only a small fraction of heritability. Maybe a part of that heritability that we detect in phenomenological twin studies is actually due to epigenetic mechanisms.

Q: What questions remain about the methods and data that you want to follow up on?
A: Overall, this was a significant effort that required over 700 microarray experiments to test nearly 100 sets of MZ and DZ twins, but it was only a pilot study. First, the twin sample—when it is split into tissue categories (white blood cells, buccal epithelial cells, and gut cells)—brings only ~20 sets per MZ or DZ twin group. Second, we used the so-called CpG island microarrays, and we interrogated only 12,000 loci in the genome. So if we add up these 12,000 loci, it would only equal about 1 percent of the genome, which is merely a tiny fraction to interrogate. The next step should be a dedicated replication effort that would investigate a much larger twin sample and use more comprehensive microarrays; ideally, we would utilize whole genome tiling microarrays. In addition, it would be particularly interesting to investigate some complex phenotypes and try to identify links between phenotypic differences in MZ and DZ co-twins vs. epigenetic differences in such co-twins.

Q: Is anything known about the difference between methylation or epigenetics in general in the nervous system versus the tissue types that you sampled? How does that play into your thinking about this? And do you have any ideas for how to surmount the fact that you’re not looking directly at the nervous system if you start thinking about this in terms of your schizophrenia or bipolar disorder research?
A: You’re absolutely right; this is a limitation. However, we have to separate basic human epigenetic studies from those in human diseases. If we want to learn about the principles of epigenetic metastability, blood, gut, or buccal cells are sufficient. In human disease epigenetic studies, unfortunately, we often cannot acquire tissue from the disease site. This directly applies to psychiatric diseases where brain samples are available only postmortem. Still, it is important to note that the non-brain tissues may contain vestiges of epigenetic changes that were inherited or occurred before tissue differentiation in embryogenesis. In our earlier studies, we detected that, despite significant epigenetic differences across tissues, epigenetic profiles from different tissues of the same individuals exhibit some degrees of similarity. Another important aspect is that the non-brain tissues may help to address the cause-effect conundrum between epigenetic changes and disease. If epigenetic abnormalities are detected in the brains of schizophrenia patients, we cannot immediately know which of these changes caused or predisposed to schizophrenia and which ones were induced by the disease or disease-associated events (e.g., compensatory mechanisms of the brain, treatment with neuroleptics). Compared to the brain, the non-brain tissues should contain fewer secondary epigenetic changes.

Q: Is there any chance that variation in DNA sequence could contribute to the differences between the monozygotic co-twins?
A: Yes, the idea that monozygotic twins are not necessarily identical in their DNA sequences is not new. The most recent observation is actually that monozygotic twins may have some copy-number variation, and there was a paper published from Jan Dumanski’s lab in the American Journal of Human Genetics last year (
Bruder et al., 2008) showing that there are some loci in monozygotic twins that exhibit copy-number variation. There are some other studies over the last 15 years demonstrating that there are some somatic mutations—trinucleotide repeats and some single-nucleotide mutations—that can potentially make monozygotic twins different. All these observations are very interesting, and I believe many more insights are still to come when sequencing of the entire human genome will become less expensive. It is early to prognosticate what proportion of phenotypic differences will be explained by DNA sequence variation or epigenetic differences. At this point all we can say is that epigenetic differences in MZ twins by far exceed the DNA sequence ones.

Comments on News and Primary Papers
Comment by:  Shiva SinghRichard O'Reilly
Submitted 2 February 2009
Posted 3 February 2009

The methylation difference between twins is clearly demonstrated using newer methods in this publication. However, conceptually it’s an old story now. A quick PubMed search for "monozygotic twins and non-identical" yielded a total of 7,653 publications. There is no doubt that the more we look, the more difference we will find between monozygotic twins. Also, monozygotic twin differences in methylation and gene expression are expected to increase with age. It is also affected by a variety of genetic and environmental factors. We have come a long way in genetic research on twins and the time has come to modify our thinking about monozygotic twins as "non-identical but closest possible" rather than as "identical." They started from a single zygote, but have diverged during development and differentiation including upbringing.

The implication of the published results is that the methylation (epigenetic) differences (in monozygotic twins) will be powerful in any genetic analysis of disease(s). Once again, it is probably more problematic than usually assumed. Also, it is particularly problematic for behavioral/psychiatric disorders including schizophrenia. The reason is multi-fold and includes the effect of (known and unknown) environment including pregnancy, upbringing, drugs, life style, food, etc. All these are known to affect DNA methylation and gene expression. As a result, they add unavoidable confounding factors to the experimental design. It does not mean that epigenetics is not involved in these diseases. Rather, directly establishing a role for methylation in schizophrenia will be challenging. A special limitation is the fact that methylation is known to be cell-type specific, and perfectly matched affected and normal (twin) human brain (region) samples for necessary experiments are problematic and methylation studies on other cell types may or may not be informative.

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Comments on Related News

Related News: SfN 2005: Nature or Nurture—Epigenetics in Neuronal Responses

Comment by:  Robert Fisher
Submitted 24 December 2005
Posted 3 January 2006

Dr. Eric Nestler at UT Southwestern Medical Center, Dallas, has long postulated addiction as a 50/50 percent split between genetic predisposition and biological changes as a result of substance abuse and the brain's accommodation to the same. I have found this particular hypothesis to be quite useful in treating addicts and alcoholics in inpatient and outpatient settings. They usually are able to easily grasp the concepts on a reasonably scientific level. This approach allows them to avoid guilt, shame, ect., that are typically strong predictors of success or failure in treatment. It is an even more successful tool in working with co-occurring Axis I disorders.

View all comments by Robert Fisher

Related News: On Again, Off Again—DNA Methylation, Genes, and Plasticity

Comment by:  David Yates
Submitted 18 April 2007
Posted 26 April 2007

Are these studies of relevance to the report from Israel that older men feed their mutations into the gene pool and this in part accounts for keeping the “schizophrenia gene” going despite poor fertility (Malaspina et al., 2002)? And might a comparison of the DNA of healthy siblings born before the mutations of an “older man” mutation with that of a sibling who got such a later mutation and developed schizophrenia reveal something of interest?

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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.


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

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