19 March 2008. A genome-wide scan for alterations in DNA methylation has found several differences in brain tissue from people with schizophrenia and bipolar disorder compared to unaffected people. The observed changes, affecting one in 200-odd genes surveyed, are consistent with the epigenetic theory of schizophrenia, which holds that the pathophysiology of schizophrenia could stem from changes in gene expression driven by aberrant chromatin structure, rather than from genetic changes in DNA sequence. The study, from the lab of Arturas Petronis and colleagues at the Center for Addiction and Mental Health in Toronto, Ontario, Canada, did not pinpoint any one or two genes, but produced a host of candidates for further study. The work appears in the March issue of the American Journal of Human Genetics.
Epigenetic influences on gene expression include methylation, acetylation, and other modifications (see SRF Current Hypothesis by Grayson and colleagues, and SRF related news story). Methylation at cytosine-guanine (CG)-rich "islands" surrounding genes is important for silencing, and is necessary to create stable configurations of gene expression in differentiated cells. A recent paper analyzing methylation at 50 genetic loci in human cerebral cortex showed that DNA methylation is dynamic over time, involves differentiated neurons, and affects a substantial portion of genes (Siegmund et al., 2007). In schizophrenia, changes in the methylation status of a few genes have been reported (see SRF related news story), but the new study is the first large-scale look at changes in methylation in the disease.
For the initial analysis, first author Jonathan Mill and colleagues extracted genomic DNA from postmortem brain tissue of 105 subjects (from the Stanley Medical Research Institute collection), of which one-third had schizophrenia, one-third had bipolar disorder, and one-third were demographically matched controls. The unmethylated fraction of DNA was enriched using methylation-sensitive restriction enzymes and hybridized to gene chip arrays covering 12,000 CpG islands, spanning the GC-rich regions around genes where methylation occurs. The relative hybridization signals indicated genes that were hyper- or hypomethylated in the different groups.
Changes in methylation were quite common between affected and unaffected subjects, and the affected genes included many involved in glutamatergic and GABAergic neurotransmission, and neuronal development. There were sex differences in methylation, but males and females with schizophrenia had similar patterns overall. The researchers singled out hypermethylation in two genes in this regard: RPP21, a component of ribonuclease P involved in tRNA maturation, and KEL, a blood group glycoprotein, whose abnormal expression has been tied to symptoms of schizophrenia. Both genes were also hypermethylated in females with bipolar disorder. However, in the bipolar group, there was no significant correlation of methylation between males and females, suggesting stronger sex-specific factors at play in that disease. No association was found between demographics and methylation at any one gene, with the exception that hypomethylation upstream of the MEK1 gene correlated with lifetime antipsychotic use in the male schizophrenia group. A network analysis of the methylation data in cases versus controls suggested that there might be a widespread epigenetic imbalance in major psychosis.
Methylation appeared to reflect lower gene expression in some cases where mRNA analysis had been done on the same Stanley collection samples for other studies. Comparing methylation data with gene expression data, the investigators found that 82 percent of loci that were hypermethylated had lower gene expression in at least one study, with one quarter showing significant downregulation across several studies. For hypomethylation, the concordance with higher gene expression was not as tight.
From the genes implicated in the chip analysis, the researchers chose 10 for a closer look at the sites of methylation. They did this by treating the DNA with sodium bisulfite, which converts normal cytosine residues to thymidine, leaving the methylated cytosines intact, and then quantifying the degree of methylation across selected regions by Pyrosequencing. The results confirmed the chip analysis in a number of loci.
Using the sequencing approach, the investigators also looked at methylation in a set of candidate genes chosen for their implication in schizophrenia. They failed to find psychosis-associated methylation differences in any of these genes, which included two, COMT and RELN, where hypomethylation had been reported before (see SRF related news story and Grayson et al., 2005). The reason for this discrepancy is unclear. Identification of epigenetic changes could be confounded by variations in the tissue samples, where distinct neuronal types have their own methylation profiles (Ruzicka et al., 2007).
These types of epigenetic studies are still in their infancy, and much remains to be done. Going forward, the authors conclude that, “The unbiased microarray approach was far more productive in identification of differentially methylated loci than was the focused candidate-gene approach; this has implications for the design of future epigenetic studies for complex disease.”—Pat McCaffrey.
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;82(3):696-711. Abstract