**3. The role of DNA methylation in the development of psychosis**

DNA methylation represents a core epigenetic mechanism that involves the covalent binding of a methyl group to the 5‐carbon position of cytosine, often leading to altered gene expression [17]. DNA methylation is influenced by stochastic events, including exposure to a variety of environmental factors, such as drug treatment [18, 19]. Epigenetic mechanisms, including DNA methylation, regulate normal cognition, neurodevelopment, and function. In addition, DNA sequence variations only explain a small proportion of the heritability of the disease. The remaining heritability, often referred to as missing heritability, could be, at least partially, explained by epigenetic changes. Interestingly, a number of animal model studies of neuro‐ developmental disorders signified that reversing the underlying molecular deficits could lead to substantial improvements in function giving hope to effective treatments even starting in adulthood [20]. These points highlight the need to further investigate the role of epigenetic signatures in the etiology and treatment of psychiatric disorders, including schizophrenia.

With this in mind, our lab has performed two sets of studies on DNA methylation in schizo‐ phrenia. The first study focused on two pairs of monozygotic twins discordant for schizo‐ phrenia and their parents to investigate differences in genome‐wide DNA methylation using a NimbleGen Methylation Promoter Microarray. Since monozygotic twins share nearly identical DNA, the study represents an ideal design to investigate the role of DNA methylation in the etiology of the disease. The genomic DNA was processed at ArrayStar (Rockville, MD, USA). Pair files were analyzed with the tiling workflow in Partek Genomics Suite version 6.6 (St. Louis, Missouri, USA). Details of the methodology have been previously described [4]. As a result, differentially methylated regions (DMRs) were identified between discordant monozygotic twins. Some of the DMRs were shared with parents of the discordant twins while others represented *de novo* methylation changes [4]. The study also reported that 27 genes were affected by DMR changes that were commonly detected in the schizophrenia‐affected member of the two discordant monozygotic pairs of unrelated families. Many of these genes were found to be a part of the histone coding gene family, which has been previously linked to the causation of schizophrenia [21–23]. Moreover, the identified genes affected by DMRs were linked to specific networks including "cell death and survival" and "cellular movement and immune cell trafficking" [4]. Interestingly, those genes and their networks have been previously associated with the etiology of schizophrenia. The findings of this particular study corrobo‐ rated the notion that DNA methylation may play a critical role in the discordance of mono‐ zygotic twins for schizophrenia. The results also shed light on the relevance of gene‐specific DNA methylation changes and on the involvement of multiple genes harboring methylation changes across specific pathways in the discordance of monozygotic twins for schizophrenia.

The second study comprised an animal model experiment investigating genome‐wide DNA methylation changes following the administration of a therapeutic dose of olanzapine in rats *in vivo*. Hippocampus and cerebellum brain regions were used and liver was included as a nonbrain tissue [5]. As a result, our study revealed that DNA methylation is not only involved in the etiology of mental health disorders, but also may be the underlying mechanism by which antipsychotic drugs function in treating the disorder. This was supported by a number of pathways significantly influenced by methylation changes. These included "nervous system development and function, tissue morphology, cellular assembly and organization," (**Fig‐ ure 3**). These findings suggested that an increase or decrease in DNA methylation of specific gene promoters, following olanzapine treatment, might decrease or increase transcriptional efficiency [37, 38], specifically in the hippocampus. The hippocampus is viewed as one of the primary sites associated with psychotic symptoms [7, 24, 25]. We also reported that the *dopamine‐DARPP32 feedback in cAMP signaling* pathway (*p* < 1.6E‐3) was the most significant pathway identified in the hippocampus region of the olanzapine‐treated rat brain. Neurons in the midbrain release dopamine, which modulates *cAmp* (cyclic adenosine 3,5‐monophosphate) production by activating dopamine receptors [1]. These results may suggest that antipsychotic effects of olanzapine involve alterations in gene‐specific methylation that would lead to dis‐ regulation of genes involved in the dopamine *DARPP32 feedback in cAmp signaling* pathway. This includes several differentially methylated genes such as *Drd1/5* and *Nos1*. It is an estab‐ lished fact that dopamine blockade leads to the progressive treatment of psychosis while its disturbance leads to the manifestation of psychosis [26]. And, all currently used antipsychotics block postsynaptic D2 receptors [27].

Schizophrenia patients either partially respond to antipsychotic drugs or do not respond at all [28]. This may be due to several factors, and one possibility is the delay in the onset of therapeutic actions partly or fully influenced by downstream effects, such as altered tran‐ scription [29, 30]. As such, differentially methylated genes involved in the dopamine‐signaling pathway may stop or reduce transcription and gene expression [17, 29, 30].

Significant hypomethylation in two CpG sites of the *FAM63B* gene in bipolar disorder patients have been recently reported [31]. Their findings plus previous hypomethylation results reported in another study involving schizophrenia patients suggest that *FAM63B* may be a common risk gene for both disorders. Although the authors reported correlation in methyla‐ tion levels at the two sites, they did not find significant association of DNA methylation with Changes in DNA Sequence and Methylation Contribute to the Predisposition of Schizophrenia: Toward... http://dx.doi.org/10.5772/65905 35

of the two discordant monozygotic pairs of unrelated families. Many of these genes were found to be a part of the histone coding gene family, which has been previously linked to the causation of schizophrenia [21–23]. Moreover, the identified genes affected by DMRs were linked to specific networks including "cell death and survival" and "cellular movement and immune cell trafficking" [4]. Interestingly, those genes and their networks have been previously associated with the etiology of schizophrenia. The findings of this particular study corrobo‐ rated the notion that DNA methylation may play a critical role in the discordance of mono‐ zygotic twins for schizophrenia. The results also shed light on the relevance of gene‐specific DNA methylation changes and on the involvement of multiple genes harboring methylation changes across specific pathways in the discordance of monozygotic twins for schizophrenia.

The second study comprised an animal model experiment investigating genome‐wide DNA methylation changes following the administration of a therapeutic dose of olanzapine in rats *in vivo*. Hippocampus and cerebellum brain regions were used and liver was included as a nonbrain tissue [5]. As a result, our study revealed that DNA methylation is not only involved in the etiology of mental health disorders, but also may be the underlying mechanism by which antipsychotic drugs function in treating the disorder. This was supported by a number of pathways significantly influenced by methylation changes. These included "nervous system development and function, tissue morphology, cellular assembly and organization," (**Fig‐ ure 3**). These findings suggested that an increase or decrease in DNA methylation of specific gene promoters, following olanzapine treatment, might decrease or increase transcriptional efficiency [37, 38], specifically in the hippocampus. The hippocampus is viewed as one of the primary sites associated with psychotic symptoms [7, 24, 25]. We also reported that the *dopamine‐DARPP32 feedback in cAMP signaling* pathway (*p* < 1.6E‐3) was the most significant pathway identified in the hippocampus region of the olanzapine‐treated rat brain. Neurons in the midbrain release dopamine, which modulates *cAmp* (cyclic adenosine 3,5‐monophosphate) production by activating dopamine receptors [1]. These results may suggest that antipsychotic effects of olanzapine involve alterations in gene‐specific methylation that would lead to dis‐ regulation of genes involved in the dopamine *DARPP32 feedback in cAmp signaling* pathway. This includes several differentially methylated genes such as *Drd1/5* and *Nos1*. It is an estab‐ lished fact that dopamine blockade leads to the progressive treatment of psychosis while its disturbance leads to the manifestation of psychosis [26]. And, all currently used antipsychotics

Schizophrenia patients either partially respond to antipsychotic drugs or do not respond at all [28]. This may be due to several factors, and one possibility is the delay in the onset of therapeutic actions partly or fully influenced by downstream effects, such as altered tran‐ scription [29, 30]. As such, differentially methylated genes involved in the dopamine‐signaling

Significant hypomethylation in two CpG sites of the *FAM63B* gene in bipolar disorder patients have been recently reported [31]. Their findings plus previous hypomethylation results reported in another study involving schizophrenia patients suggest that *FAM63B* may be a common risk gene for both disorders. Although the authors reported correlation in methyla‐ tion levels at the two sites, they did not find significant association of DNA methylation with

pathway may stop or reduce transcription and gene expression [17, 29, 30].

block postsynaptic D2 receptors [27].

34 Schizophrenia Treatment - The New Facets

**Figure 3.** (A) Nervous system development and function, tissue morphology, cellular assembly and organization. (B) Metabolic disease, tissue morphology, endocrine system disorders. Genes shaded in gray were affected by changes in promoter methylation [5].

nearby SNPs, which may further corroborate the biological significance of identifying epige‐ netic signatures in addition to conducting genome‐wide association analyses. On their own, association analyses will not always reveal risk variants or genes due to their limitations such as low statistical power or the presence of genotyping error as well as the detection of false positives.

Different brain regions as well as a variety of cell types are known to have different epige‐ netic signatures, and depending on the subpopulations analyzed, specific cell types may even show different epigenetic signatures within their own subpopulation [32, 33]. These reported differences in epigenetic signatures of different brain regions and our observa‐ tions of significant differences in DNA methylation patterns of hippocampus and cerebel‐ lum in a rat model study, reflect the possibility that these epigenetic signatures may play a role in regulating gene expression and thereby causing psychiatric disorders including schizophrenia.

Several CpGs have been reported to show significant differences in DNA methylation levels in psychosis cases [34]. These results shed light on the significance of epigenetic signatures in the causes and treatment of mental health disorders. Our previous studies revealed brain tissue‐specific DNA methylation changes [5]. As a result, scientists are now advising for caution when interpreting the findings of DNA methylation differences in schizophrenia affected and healthy subjects using peripheral tissues, including blood samples [35].

As we noted in our previous reports, olanzapine caused an increase or a decrease in methyl‐ ation of genes previously implicated in schizophrenia, which may reflect the fact that olanza‐ pine could result in the recovery of psychiatric symptoms via mechanisms involving DNA methylation. Among the genes that showed a decrease in methylation in hippocampus is *Map6*, which is implicated in schizophrenia [36], and involved in molecular transport, nervous system development, and function [5]. This implies that methylation may serve an interme‐ diary role whose actual effect is realized through gene expression.

Apart from the involvement of DNA methylation in the treatment of schizophrenia via antipsychotic administration, methylation changes may also affect genes and pathways that reflect the side effects of the drugs. In a genome‐wide assessment, our studies showed methylation changes in several genes and pathways that may alter metabolomics leading to the efficacy as well as side effects of olanzapine. The side effects were reflected by significant increases in body weight gain and a pathway affecting metabolic disorders. Interestingly, genetic variations in various genes including *BDNF* have been implicated in antipsychotic‐ induced weight gain [37]. However, the relationship between sequence variations and methylation changes in leading to the predisposition of individuals to the disease, their role in the efficacy of antipsychotic treatment, and also their role in the side effects of the drugs remains to be investigated.

It is an established fact that genomic imprinting is an epigenetic phenomenon by which certain genes are expressed in a parent‐of‐origin specific manner [38]. Also, X‐chromosome inactivation invariably involves epigenetic phenomenon [39, 40]. The genomic distributions of epimutations play an important role in their effects on the disorder. In particular, we would like to emphasize that not all observed epigenetic changes in the genome play a role in the regulation of gene expression. Although most epimutations located in nonpromoter regions do not often lead to changes in gene expression, epigenetic signatures in the pro‐ moter regions are often associated with regulation of gene expressions [29, 41]. Interesting‐ ly, long‐lasting alterations in DNA methylation and their effect on neurodevelopmental disorders have been reported [42]. However, changes in DNA methylation needs to be in‐ terpreted with caution, as methylation and its function are context‐dependent [43]. More importantly, effects of epimutations on neurodevelopmental disorders are tissue‐specific, cell‐type, and organ specific [44, 45]. Interestingly, our lab reported tissue‐specific changes in promoter DNA methylation of several psychosis related genes using hippocampus, cere‐ bellum, and liver samples [5].

nearby SNPs, which may further corroborate the biological significance of identifying epige‐ netic signatures in addition to conducting genome‐wide association analyses. On their own, association analyses will not always reveal risk variants or genes due to their limitations such as low statistical power or the presence of genotyping error as well as the detection of false

Different brain regions as well as a variety of cell types are known to have different epige‐ netic signatures, and depending on the subpopulations analyzed, specific cell types may even show different epigenetic signatures within their own subpopulation [32, 33]. These reported differences in epigenetic signatures of different brain regions and our observa‐ tions of significant differences in DNA methylation patterns of hippocampus and cerebel‐ lum in a rat model study, reflect the possibility that these epigenetic signatures may play a role in regulating gene expression and thereby causing psychiatric disorders including

Several CpGs have been reported to show significant differences in DNA methylation levels in psychosis cases [34]. These results shed light on the significance of epigenetic signatures in the causes and treatment of mental health disorders. Our previous studies revealed brain tissue‐specific DNA methylation changes [5]. As a result, scientists are now advising for caution when interpreting the findings of DNA methylation differences in schizophrenia

As we noted in our previous reports, olanzapine caused an increase or a decrease in methyl‐ ation of genes previously implicated in schizophrenia, which may reflect the fact that olanza‐ pine could result in the recovery of psychiatric symptoms via mechanisms involving DNA methylation. Among the genes that showed a decrease in methylation in hippocampus is *Map6*, which is implicated in schizophrenia [36], and involved in molecular transport, nervous system development, and function [5]. This implies that methylation may serve an interme‐

Apart from the involvement of DNA methylation in the treatment of schizophrenia via antipsychotic administration, methylation changes may also affect genes and pathways that reflect the side effects of the drugs. In a genome‐wide assessment, our studies showed methylation changes in several genes and pathways that may alter metabolomics leading to the efficacy as well as side effects of olanzapine. The side effects were reflected by significant increases in body weight gain and a pathway affecting metabolic disorders. Interestingly, genetic variations in various genes including *BDNF* have been implicated in antipsychotic‐ induced weight gain [37]. However, the relationship between sequence variations and methylation changes in leading to the predisposition of individuals to the disease, their role in the efficacy of antipsychotic treatment, and also their role in the side effects of the drugs

It is an established fact that genomic imprinting is an epigenetic phenomenon by which certain genes are expressed in a parent‐of‐origin specific manner [38]. Also, X‐chromosome inactivation invariably involves epigenetic phenomenon [39, 40]. The genomic distributions of epimutations play an important role in their effects on the disorder. In particular, we

affected and healthy subjects using peripheral tissues, including blood samples [35].

diary role whose actual effect is realized through gene expression.

positives.

36 Schizophrenia Treatment - The New Facets

schizophrenia.

remains to be investigated.

Overall, the results from our lab and elsewhere suggest that aberrant DNA methylation in a set of candidate genes may be involved in mental disorders, including schizophrenia [46]. Also, it may explain the therapeutic efficacy, side effects, and individual specificity of responses to antipsychotic treatments. They may result from changes in tissue‐specific DNA methylation in a set of genes [5, 13, 47, 48]. However, DNA methylation changes in mental health patients and their effects on the expression of psychosis relevant genes as well as the development of psychotic symptoms require attention in future studies. Also, effects of DNA methylation on the expression of specific sets of genes leading to the development of schizophrenia, requires further investigation. To this effect, the use of endophenotypes (intermediate phenotypes that are quantifiable traits of the disease) may help facilitate the investigation of the underlying biological basis of schizophrenia. The United States Food and Drug Administration has accepted endophenotypes as therapeutic treatment targets [49]. Further, our study based on two sets of studies (a rat model investigating effects of olanzapine on DNA methylation of brain regions, and monozygotic twins discordant for schizophrenia) revealed genes and gene networks commonly affected in the two sets of studies. The findings reflect the fact that a considerable portion of the observed methylation changes are likely to be caused by antipsy‐ chotic drugs in both studies [50]. Also, it is likely that some of the methylation changes seen can be attributed to the underlying factors that predispose patients to the disorder. Further studies are still needed to confirm the role of methylation changes in the etiology and patho‐ physiology of the disease.
