Hamid Mostafavi Abdolmaleky

Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: A preliminary report

DNA methylation changes could provide a mechanism for DNA plasticity and dynamism for short‐term adaptation, enabling a type of cell memory to register cellular history under different environmental conditions. Some environmental insults may also result in pathological methylation with corresponding alteration of gene expression patterns. Evidence from several studies has suggested that in schizophrenia and bipolar disorder, mRNA of the reelin gene (RELN), which encodes a protein necessary for neuronal migration, axonal branching, synaptogenesis, and cell signaling, is severely reduced in post‐mortem brains. Therefore, we investigated the methylation status of the RELN promoter region in schizophrenic patients and normal controls as a potential mechanism for down regulation of its expression. Ten post‐mortem frontal lobe brain samples from male schizophrenic patients and normal controls were obtained from the Harvard Brain Tissue Resources Center. DNA was extracted using a standard phenol–chloroform DNA extraction protocol. To evaluate differences between patients and controls, we applied methylation specific PCR (MSP) using primers localized to CpG islands flanking a potential cyclic AMP response element (CRE) and a stimulating protein‐1 (SP1) binding site located in the promoter region. For each sample, DNA extraction, bisulfite treatment, and MSP were independently repeated at least four times to accurately determine the methylation status of the target region. Forty‐three PCR trials were performed on the test and control samples. MSP analysis of the RELN promoter revealed an unmethylated signal in all reactions (43 of 43) using DNA from the frontal brain tissue, derived from either the schizophrenic patients or normal controls indicating that this region of the RELN promoter is predominantly unmethylated. However, we observed a distinct methylated signal in 73% of the trials (16 of 22) in schizophrenic patients compared with 24% (5 of 21) of controls. Thus, the hypermethylation of the CpG islands flanking a CRE and SP1 binding site observed at a significantly higher level (t = −5.07, P = 0.001) may provide a mechanism for the decreased RELN expression, frequently observed in post‐mortem brains of schizophrenic patients. We also found an inverse relationship between the level of DNA methylation using MSP analysis and the expression of the RELN gene using semi‐quantitative RT‐PCR. Despite the small sample size, these studies indicate that promoter hypermethylation of the RELN gene could be a significant contributor in effecting epigenetic alterations and provides a molecular basis for the RELN gene hypoactivity in schizophrenia. Further studies with a larger sample set would be required to validate these preliminary observations.

Methylomics in psychiatry: Modulation of gene-environment interactions may be through DNA methylation.

Fine‐tuning of neuronal connections during development is regulated through environmental interactions. Some fine‐tuning occurs through changes in gene expression and/or epigenetic gene‐specific DNA methylation states. DNA methylation occurs by transfer of a methyl group from S‐adenosyl methionine to cytosine residues in the dinucleotide sequence CpG. Although CpG sequences spread throughout the genome are usually heavily methylated, those occurring in CpG islands in the promoter regions of genes are less methylated. In most cases, the extent of DNA methylation correlates with the extent of gene inactivation. Other known epigenetic mechanisms include histone deacetylation and chromatin remodeling, RNA inhibition, RNA modification, and DNA rearrangement. Exposure memory expressed as epigenetic DNA modifications allows genomic plasticity and short‐term adaptation of each generation to their environment. Environmental factors that affect DNA methylation include diet, proteins, drugs, and hormones. Induced methylation changes may produce altered gene response upon subsequent hormonal stimulation. The gene‐specific DNA methylation state may be preserved upon transmission through mitosis and meiosis. An increasing amount of data implicates a role for DNA methylation in multi‐factorial psychiatric disorders. For example, l‐methionine treatment can exacerbate psychosis; while valproate, a drug producing hypomethylated DNA, reduces such symptoms. Hypermethylation of the promoter region of the RELN gene correlates with reduced gene expression. This genes protein Reelin, which is necessary for neuronal migration and synaptogenesis, is reduced in schizophrenia and bipolar disorder, suggesting hypermethylation of the promoter region in these disorders. Some evidence implicates methylation of the promoter regions of the DRD2 and HTR2A genes in schizophrenia and mood disorders as well. DNA methylation usually increases with age, although hypomethylation of the promoter region of the amyloid A4 precursor gene during aging may play a role in Alzheimers disease. More studies are needed to define the role of methylomics and other epigenetic phenomena in the nervous system.

Meta-analysis of association between the T102C polymorphism of the 5HT2a receptor gene and schizophrenia

A meta-analysis of whole-genome linkage scans confirmed linkage between schizophrenia and markers on the long arm of chromosome 13. The gene HTR2A, which codes for the 5HT2a receptor, is located in this area. The T102C single nucleotide polymorphism of HTR2A has been the subject of much research. The production of the C-allele form of HTR2A is significantly less than that of the T-allele form in normal controls and schizophrenic patients. Although the association of schizophrenia with the C allele of HTR2A was confirmed by a meta-analysis 5 years ago, there was a continuous debate because negative findings were also considerable, which may have been due to ethnic differences in association. We performed another meta-analysis, since the number of available studies of this association has recently doubled. In the meta-analysis of 31 case-control association studies, we found a significant association between the C allele of the T102C polymorphism and schizophrenia, which was more pronounced in European samples than in the entire sample. We found significant heterogeneity in the allele-wise analysis (C vs. T) and homozygous genotype-wise analysis (CC vs. TT), both of which were at least partially explained by differences between samples from Asian and European countries. In East Asian countries, there was not a significant association with the C allele or CC homozygosity, indicating strong genetic differences and noncombinability of data between European and East Asian populations. Interestingly, the frequency of the T allele was much higher in East Asian patients and controls (59.5% and 57.5%, respectively) than in European patients and controls (40% and 43.5%, respectively). In five family-based association studies, we did not find significant evidence for association of the C allele with schizophrenia; yet, the pooled OR was 1.3 (95% CI=0.9-1.8, z=1.47, p=0.14), which is consistent with the results of the case-control studies. The effects of other genes, environmental effects on DNA methylation, or different methods of classification may be the causes for such heterogeneity, but more study in this area is needed.

Genetics and epigenetics in major psychiatric disorders: dilemmas, achievements, applications, and future scope.

No specific gene has been identified for any major psychiatric disorder, including schizophrenia, in spite of strong evidence supporting a genetic basis for these complex and devastating disorders. There are several likely reasons for this failure, ranging from poor study design with low statistical power to genetic mechanisms such as polygenic inheritance, epigenetic interactions, and pleiotropy. Most study designs currently in use are inadequate to uncover these mechanisms. However, to date, genetic studies have provided some valuable insight into the causes and potential therapies for psychiatric disorders.There is a growing body of evidence suggesting that the understanding of the genetic etiology of psychiatric illnesses, including schizophrenia, will be more successful with integrative approaches considering both genetic and epigenetic factors. For example, several genes including those encoding dopamine receptors (DRD2, DRD3, and DRD4), serotonin receptor 2A (HTR2A) and catechol-O-methyltransferase (COMT) have been implicated in the etiology of schizophrenia and related disorders through meta-analyses and large, multicenter studies. There is also growing evidence for the role of DRD1, NMDA receptor genes (GRIN1, GRIN2A, GRIN2B), brain-derived neurotrophic factor (BDNF), and dopamine transporter (SLC6A3) in both schizophrenia and bipolar disorder. Recent studies have indicated that epigenetic modification of reelin (RELN), BDNF, and the DRD2 promoters confer susceptibility to clinical psychiatric conditions.Pharmacologic therapy of psychiatric disorders will likely be more effective once the molecular pathogenesis is known. For example, the hypoactive alleles of DRD2 and the hyperactive alleles of COMT, which degrade the dopamine in the synaptic cleft, are associated with schizophrenia. It is likely that insufficient dopaminergic transmission in the frontal lobe plays a role in the development of negative symptoms associated with this disorder. Antipsychotic therapies with a partial dopamine D2 receptor agonist effect may be a plausible alternative to current therapies, and would be effective in symptom reduction in psychotic individuals. It is also possible that therapies employing dopamine D1/D2 receptor agonists or COMT inhibitors will be beneficial for patients with negative symptoms in schizophrenia and bipolar disorder. The complex etiology of schizophrenia, and other psychiatric disorders, warrants the consideration of both genetic and epigenetic systems and the careful design of experiments to illumine the genetic mechanisms conferring liability for these disorders and the benefit of existing and new therapies.

Hypomethylation of the serotonin receptor type-2A Gene (HTR2A) at T102C polymorphic site in DNA derived from the saliva of patients with schizophrenia and bipolar disorder.

Several lines of evidence indicate that dysfunction of serotonin signaling and HTR2A receptor are involved in the pathogenesis of schizophrenia (SCZ) and bipolar disorder (BD). DNA methylation of HTR2A at T102C polymorphic site influences HTR2A expression and aberrant DNA methylation of HTR2A promoter was reported in postmortem brain of patients with SCZ and BD. Hypothesizing that the brains epigenetic alteration of HTR2A may also exist in peripheral tissues that can be used as a diagnostic/therapeutic biomarker, we analyzed HTR2A promoter DNA methylation in DNA extracted from the saliva of patients with SCZ and BD, and their first degree relatives versus normal controls. Bisulfite sequencing was used to screen DNA methylation status of the HTR2A promoter CpGs and qMSP was used to quantify the degree of cytosine methylation at differentially methylated sites. Most of the cytosines of the HTR2A promoter were unmethylated. However, CpGs of the −1438A/G polymorphism site, −1420 and −1223 were >95% methylated. The CpG at T102C polymorphic site and neighboring CpGs were ∼70% methylated both in the patients and controls. qMSP analysis revealed that the cytosine of the T102C polymorphic site was significantly hypo‐methylated in SCZ, BD, and their first degree relatives compared to the controls. Cytosine methylation of HTR2A at T102C polymorphic site in DNA derived from the saliva can potentially be used as a diagnostic, prognostic, and/or therapeutic biomarker in SCZ and BD. However, these preliminary observations need to be replicated in other populations with a larger sample size to be considered for clinical applications.

An update on the epigenetics of psychotic diseases and autism

The examination of potential roles of epigenetic alterations in the pathogenesis of psychotic diseases have become an essential alternative in recent years as genetic studies alone are yet to uncover major gene(s) for psychosis. Here, we describe the current state of knowledge from the gene-specific and genome-wide studies of postmortem brain and blood cells indicating that aberrant DNA methylation, histone modifications and dysregulation of micro-RNAs are linked to the pathogenesis of mental diseases. There is also strong evidence supporting that all classes of psychiatric drugs modulate diverse features of the epigenome. While comprehensive environmental and genetic/epigenetic studies are uncovering the origins, and the key genes/pathways affected in psychotic diseases, characterizing the epigenetic effects of psychiatric drugs may help to design novel therapies in psychiatry.

Aberrant activation of γ-catenin promotes genomic instability and oncogenic effects during tumor progression

γ-catenin (plakoglobin) exists in cells either as a component of adherens junctions, along with β-catenin and α-catenin, or in association with desmoplakin in desmosomes, which are in turn coupled to the cytoskeleton linking to the plasma membrane. Although γ-catenin overexpression is observed in many cancers, the molecular basis of its contribution to tumor progression remains unclear. In this study, we examined γ-catenin overexpression-mediated effects leading to altered regulation of effector genes such as PTTG and c-Myc, as well as differential activation of signaling pathways. We found that overexpression of γ-catenin caused: (1) a reduction in E-cadherin and corresponding increase in vimentin levels concomitant with increased cell mobility and migration; (2) enhancement in the levels of phosphorylated Akt and Erk in the presence of EGF; and (3) an increase in PTTG and c-Myc protein levels, which are likely to accelerate chromosomal instability and uncontrolled proliferation, respectively, in the affected cells. These effects resulting from overexpression of γ-catenin were further validated in converse experiments with the aid of siRNA knockdown of the endogenous γ-catenin gene. In conclusion, our studies provide a molecular basis for the promotion of genomic instability and the oncogenic effects due to overexpression of γ-catenin in human cancer.

Epigenetic and pharmacoepigenomic studies of major psychoses and potentials for therapeutics

Individuals with neuropsychiatric diseases have epigenetic programming disturbances, both in the brain, which is the primary affected organ, and in secondary tissues. Epigenetic modulations are molecular modifications made to DNA, RNA and proteins that fine-tune genotype into phenotype and do not include DNA base changes. For instance, gene-expression modulation is linked to epigenetic codes in chromatin that consist of post-replication DNA methylation and histone protein modifications (e.g., methylation, acetylation and so on), particularly in gene-promoter regions. Epigenetic coding is modulated globally, and in a gene-specific manner by environmental exposures that include nutrition, toxins, drugs and so on. Analysis of epigenetic aberrations in diseases helps to identify dysfunctional genes and pathways, establish more robust cause-effect relationships than genetic studies alone, and identify new pharmaceutical targets and drugs, including nucleic acid reagents such as inhibitory RNAs. The emerging science of pharmacoepigenomics can impact the treatment of psychiatric and other complex diseases. In fact, some therapeutics now in use target epigenetic programming. In the near future, epigenetic interventions should help stabilize affected individuals and lead to prevention strategies.

hBub1 negatively regulates p53 mediated early cell death upon mitotic checkpoint activation

Our previous studies showed that the depletion of the outer kinetochore protein hBub1 upon activation of spindle assembly checkpoint (SAC) primarily triggers early cell death mediated by p53 rather than aneuploidy. Here, we report that phosphorylation of p53 at the Ser 37 is critical for its proapoptotic activity upon SAC activation. Furthermore, we show that p53 physically interacts with hBub1 at kinetochores in response to mitotic spindle damage suggesting a direct role for hBub1 in the suppression of p53 mediated cell death. This observation is further substantiated by the inhibition of p53 mediated transactivation of the proapoptotic target genes, PUMA and BAX, by hBub1 in SAC activated cells. In summary, our data from these and our previous studies strongly suggest that in response to SAC activation, hBub1 acts as a negative regulator of p53 mediated early cell death in a novel checkpoint pathway. On the translational medicine front, it is tempting to speculate that by disabling the hBub1 in p53 proficient cancer cells, apoptosis may be induced as a therapeutic approach to eradicate the tumor cells.

Microbiome, inflammation, epigenetic alterations, and mental diseases

Major mental diseases such as autism, bipolar disorder, schizophrenia, and major depressive disorder are debilitating illnesses with complex etiologies. Recent findings show that the onset and development of these illnesses cannot be well described by the one‐gene; one‐disease approach. Instead, their clinical presentation is thought to result from the regulative interplay of a large number of genes. Even though the involvement of many genes are likely, up regulating and activation or down regulation and silencing of these genes by the environmental factors play a crucial role in contributing to their pathogenesis. Much of this interplay may be moderated by epigenetic changes. Similar to genetic mutations, epigenetic modifications such as DNA methylation, histone modifications, and RNA interference can influence gene expression and therefore may cause behavioral and neuronal changes observed in mental disorders. Environmental factors such as diet, gut microbiota, and infections have significant role in these epigenetic modifications. Studies show that bioactive nutrients and gut microbiota can alter either DNA methylation and histone signatures through a variety of mechanisms. Indeed, microbes within the human gut may play a significant role in the regulation of various elements of “gut–brain axis,” via their influence on inflammatory cytokines and production of antimicrobial peptides that affect the epigenome through their involvement in generating short chain fatty acids, vitamin synthesis, and nutrient absorption. In addition, they may participate in‐gut production of many common neurotransmitters. In this review we will consider the potential interactions of diet, gastrointestinal microbiome, inflammation, and epigenetic alterations in psychiatric disorders.