One of the main epigenetic mechanisms is DNA methylation. It mostly occurs in CpG islands in the promoters by addition of a methyl group to a cytosine that may inhibit gene expression. It is known ...that many developmental disorders are characterized by alterations in epigenetic mechanisms. Moreover, studies in animal models of neuropsychiatric disorders have shown that epigenetics may play an important role in pathogenesis and therapy. The aim of this study is to investigate the effect of drugs used in different psychiatric diseases including Tourette Syndrome (TS) on DNA methylation to find epigenetic targets that may influence the efficacy of treatment.
Treatment: Wistar Kyoto rats were chronically treated from post-natal day (PND) 35 to PND50 by intraperitoneal injection of either aripiprazole or riluzole or vehicle. Metabolite changes during the treatment were quantified by magnetic resonance spectroscopy in the striatum and prefrontal cortex.
Samples: DNA isolated from striatum and prefrontal cortex was used for methylation studies. Reduced Representation Bisulfite Sequencing (RRBS): RRBS is a high-throughput technique used to analyze genome-wide methylation profiles at the single nucleotide level. It provides information of the most relevant genomic regions which are likely to influence gene expression. We analyzed striatum and prefrontal cortex of 2 rats in each group treated with aripiprazole (a dopaminergic neuroleptic) or riluzole (a glutamatergic anxiolytic) or vehicle in order to identify DNA methylation changes due to these drug treatments.
Mass-Spectrometry: The analysis was performed by LC-MS/MS on striatum and prefrontal cortex in order to quantify the amount of 5-methyl-Cytosine (mC) and 5-HydroxyMethylCytosine (5hmC) after drug treatment.
Mass-Spectrometry: LC-MS/MS showed significant differences in 5hmC levels in prefrontal cortex after treatment with aripiprazole and riluzole. The same trend was also observed for mC levels. We found no differences in methylation or hydroxy-methylation levels in striatal samples.
RRBS: we observed differences in overall methylation levels between treatment using the two drugs and the vehicle. We identified differentially methylated CpG sites (DMS) and characterized their distribution across the genome (eg. promoter, intron, exon, UTR, etc.).
Treatment with riluzole led to identification of 156 DMS in striatum and 346 DMS in prefrontal cortex. Treatment with aripiprazole yielded 117 DMS in striatum and 466 DMS in prefrontal cortex. The identified CpG sites were annotated, a candidate gene list was created and pathway analysis was also performed on these genes. Interestingly, most of the changes occurred in intergenic regions far from TSS suggesting that enhancers are the regions most prone to undergo methylation changes. Moreover, several genes (eg. OXTR, DRD2, etc.) have been associated with brain disorders or functions.
The known functions of implicated genes suggest that some of the observed epigenetic changes might underlie the amelioration of symptoms by these drugs and some may account for certain adverse effects. Data on differential DNA methylation will be compared with imaging data on the respective brain regions. The results give insights into the mechanism of action of aripiprazole and riluzole as well as the side effects, not just in TS, but also in a broader context regarding these psychiatric medications.
Tourette Syndrome (TS) is a neurodevelopmental disorder that presents early in childhood and is marked by the appearance of multiple involuntary motor tics and at least one vocal tic. It presents ...high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). Despite a strong genetic contribution, the molecular mechanisms behind TS are still uncertain, although multiple lines of evidence suggest involvement of specific candidate genes and corresponding epigenetic mechanisms via miRNA regulators.
To date only a few genetic findings have been replicated in TS. Among these the nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene has been recently suggested as candidate susceptibility gene. CHRNA7 is known to regulate a wide variety of developmental and secretory functions, however, the mechanism of its transcriptional regulation is still unclear 1. Another recent promising finding in TS genetic research is Netrin 4 (NTN4), which belongs to a family of extracellular proteins that direct axon outgrowth and guidance 2. Several microRNAs have already been associated with neuropsychiatric disorders, but most importantly a few miRNA have also been associated with Tourette Syndrome 3,4.Our goal was to investigate the role of selected candidate genes in a case-control setup along with functional validation involving miRNA regulation in the possible involvement in TS pathogenesis through analysis of 3’UTR regions.
An OpenArray platform (TaqMan® OpenArray® Genotyping System) was used for the case-control analysis of TS patients (N=564) and healthy controls. Starting from a list of candidate genes previously indicated as possible risk factors for TS, we screened for presence of SNPs in the 3’UTR regulatory regions. This analysis (data retrieved by mirSNP and polymiRTS databases) led to the identification of 32 SNPs which were predicted to change the seed sequence of in silico proposed miRNAs. To proof this concept, we performed functional validation study using a luciferase assay reporter containing 3’ UTR regions of CHRNA7 and NTN4 transfected with their predicted miRNA. Specifically, miRNA-106b and miRNA-198b were identified as the most potent miRNA candidates for regulation of CHRNA7 and NTN4 gene expression. SKNF1 and HEK human cell lines were co-transfected using lipofectamine with the luciferase reporter-3’ UTR construct of CHRNA7 and NTN4 (SwitchGear Genomics) and the corresponding putative miRNAs (miR-106b, miR-198b) along with non-targeting control miRNAs (miR-196b, miR-641b) in the functional validation studies.
In order to increase our understanding of the underlying genetic and epigenetic mechanisms of TS, we aimed to study the possible miRNA regulation processes in TS-related genes, which would help not only to better understand the full genetic architecture of this disorder but also to determine how miRNAs contribute to the complexity of gene regulation in the development of disease.
OpenArray analysis identified significant differences among the selected candidates genes (LHX6, iMMP2L) confirming the implication of those genes in TS etiology. In the luciferase assays we characterized the regulatory effect of the predicted miRNAs on the expression candidate genes in a concentration-dependent manner, which showed up to 5-fold change in the relative gene expression levels in case of CHRNA7 and NTN4 thereby proving the concept of our study.
Rapid molecular diagnosis of 21‐hydroxylase deficiency by detecting the most common mutation in the 21‐hydroxylase gene is presented using primer extension and capillary electrophoresis with a ...polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5‐labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.
Objective: To examine ADHD symptom persistence and subtype stability among substance use disorder (SUD) treatment seekers. Method: In all, 1,276 adult SUD treatment seekers were assessed for ...childhood and adult ADHD using Conners’ Adult ADHD Diagnostic Interview for Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV; CAADID). A total of 290 (22.7%) participants met CAADID criteria for childhood ADHD and comprise the current study sample. Results: Childhood ADHD persisted into adulthood in 72.8% (n = 211) of cases. ADHD persistence was significantly associated with a family history of ADHD, and the presence of conduct disorder and antisocial personality disorder. The combined subtype was the most stable into adulthood (78.6%) and this stability was significantly associated with conduct disorder and past treatment of ADHD. Conclusion: ADHD is highly prevalent and persistent among SUD treatment seekers and is associated with the more severe phenotype that is also less likely to remit. Routine screening and follow-up assessment for ADHD is indicated to enhance treatment management and outcomes.
Objectives
Most of the addiction studies focus on very specific aspects of addictions, often with contradictory results, and integrated studies are quite rare. Experimental studies comparing ...underlying mechanisms of addictions and analyzing data from an integrative psychological and genetic perspective are almost nonexistent. The aim of the present paper is to describe the research protocol of the Psychological and Genetic Factors of Addictive Behaviors (PGA) study, which applies an integrative approach to understanding the acquisition, development, and maintenance of addictive behaviors.
Methods
A wide‐spectrum national study was carried out. Data were collected from 3,003 adolescents. Addictions to both psychoactive substances and behaviors were thoroughly assessed via psychometrically robust scales, which also included assessment related to a wide range of related psychological dimensions. Additionally, a DNA sample was also collected from participants.
Results
The paper presents the detailed methodology of the PGA study. Data collection procedures, instrumentation, and the analytical approach used to attain the research objectives are described.
Conclusions
Future plans, along with potential contributions of the PGA study, are also discussed. It is envisaged that the study will provide a unique opportunity to test possible mechanisms and causal pathways mediating the associations of genetic factors, psychological characteristics, and addictions.
Background and aims. Some form of gambling can be observed in nearly every society, as the gratification felt upon winning in uncertain conditions is universal. A culturally distinct form of ...gambling, associated with a traditional sporting event of archery known as “teer,” is innate to the province of Meghalaya, India. The objective of this study was to find genetic variants underlying this unique form of behavioral addiction. To better understand game-based gambling, we studied genetic variants related to dopaminergic pathways and other genes previously linked to various psychological disorders. Methods. This study was carried out on a sample of 196 Indo-Aryan adults from Shillong, Meghalaya. Genotyping of glial cell line-derived neurotrophic factor (GDNF) polymorphisms was carried out using real-time PCR. We further investigated 32 single nucleotide polymorphisms located in the 3′ UTR of additional genes of interest using an OpenArray® real-time PCR platform. Results. Case–control analysis revealed a significant association between GDNF variant rs2973033 (p = .00864, χ2 = 13.132, df = 2) and contactin-associated protein-like 2 (CNTNAP2) variant rs2530311 (p = .0448, χ2 = 13.132, df = 2) with gambling. Discussion and conclusions. Association of the GDNF gene with gambling could be attributed to its involvement in the development and survival of dopaminergic neurons. Our result is in good agreement with previous data indicating the role of GDNF in certain substance addictions. Several rare variants in the CNTNAP2 gene were also implicated in alcohol addiction in a previous study. This pilot study provides further support for the role of GDNF and CNTNAP2 in addiction behaviors.
Tourette syndrome (TS) is a neuropsychiatric disorder of complex genetic architecture involving multiple interacting genes. Here, we sought to elucidate the pathways that underlie the neurobiology of ...the disorder through genome-wide analysis. We analyzed genome-wide genotypic data of 3581 individuals with TS and 7682 ancestry-matched controls and investigated associations of TS with sets of genes that are expressed in particular cell types and operate in specific neuronal and glial functions. We employed a self-contained, set-based association method (SBA) as well as a competitive gene set method (MAGMA) using individual-level genotype data to perform a comprehensive investigation of the biological background of TS. Our SBA analysis identified three significant gene sets after Bonferroni correction, implicating ligand-gated ion channel signaling, lymphocytic, and cell adhesion and transsynaptic signaling processes. MAGMA analysis further supported the involvement of the cell adhesion and trans-synaptic signaling gene set. The lymphocytic gene set was driven by variants in FLT3, raising an intriguing hypothesis for the involvement of a neuroinflammatory element in TS pathogenesis. The indications of involvement of ligand-gated ion channel signaling reinforce the role of GABA in TS, while the association of cell adhesion and trans-synaptic signaling gene set provides additional support for the role of adhesion molecules in neuropsychiatric disorders. This study reinforces previous findings but also provides new insights into the neurobiology of TS.
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