Arturas Petronis

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

The origin of schizophrenia: genetic thesis, epigenetic antithesis, and resolving synthesis.

Traditionally, it has been thought that schizophrenia results from the interaction of predisposing genes and hazardous environmental factors. In this article, the paradigm of genes plus environment is challenged, and a new interpretation is presented, in which the emphasis on DNA sequence variation is shared with epigenetic misregulation as a critical etiopathogenic factor. Partial epigenetic stability (metastability) of gene regulation is consistent with various nonmendelian irregularities of schizophrenia, such as the presence of clinically indistinguishable sporadic and familial cases, discordance of monozygotic twins, coincidence of peaks of susceptibility with major endocrine rearrangements, and fluctuating course of disease severity, among others. It is also suggested that stochastic epigenetic events might account for a substantial portion of phenotypic variance, which traditionally has been ascribed to environmental effects. This theoretic essay is constructed according to the principle of Hegelian dialectic reasoning (thesis-antithesis-synthesis), which serves the goal of showing that the best outcome of molecular genetic studies in schizophrenia (and perhaps other complex diseases) can be expected when components that effect chromatin structure and gene regulation are taken into account and investigated comprehensively.

The genes for major psychosis: aberrant sequence or regulation?

A number of recent clinical and molecular observations in major psychosis indicate that epigenetic factors may be operational in the origin of major mental illness. This article further develops the idea that epigenetic factors may play an etiopathogenic role in schizophrenia and bipolar affective disorder. The putative role of epigenetic factors is shown by the epigenetic interpretation of genetic association studies of the genes for serotonin 2A (HTR2A) and the dopamine D3 (DRD3) receptors in schizophrenia. The idea of epigenetic polymorphism of genetic alleles is introduced, and it is argued that epigenetic variation may explain a number of controversial and unclear findings in allelic and genotypic association studies of HTR2A and DRD3. In linkage analyses of multiplex families with bipolar affective disorder (BPAD), different loci on chromosome 18 indicated co-segregation of alleles of one parental sex with the disease phenotype, and this finding implies that the epigenetic mechanism of genomic imprinting may be involved. Evidence for genomic imprinting provides the background for epigenetic cloning of BPAD risk factors by searching for differentially modified genes on chromosome 18. Finally, epigenetic studies could be relevant to the better understanding of the molecular action of antipsychotic medications. In addition to this, if epimutations are detected in major psychosis, epigenetic treatment directed at correction of epigenetic status of a specific brain gene may eventually be developed.

Lack of association between serotonin-2A receptor gene (HTR2A) polymorphisms and tardive dyskinesia in schizophrenia

Tardive dyskinesia (TD) is a disabling neurological side effect associated with long-term treatment with typical antipsychotics. Family studies and animal models lend evidence for hereditary predisposition to TD. The newer atypical antipsychotics pose a minimal risk for TD which is in part attributed to their ability to block the serotonin-2A (5-HT2A) receptor. 5-HT2A receptors were also identified in the basal ganglia; a brain region that plays a critical role in antipsychotic-induced movement disorders. We tested the significance of variation in the 5-HT2A receptor gene (HTR2A) in relation to the TD phenotype. Three polymorphisms in HTR2A, one silent (C102T), one that alters the amino acid sequence (his452tyr) and one in the promoter region (A-1437G) were investigated in 136 patients refractory or intolerant to treatment with typical antipsychotics and with a DSM-IIIR diagnosis of schizophrenia. We did not find any significant difference in allele, genotype or haplotype frequencies of polymorphisms in HTR2A among patients with or without TD (Pu2009>u20090.05). Further analysis using the ANCOVA statistic with a continuous measure of the TD phenotype (Abnormal Involuntary Movement Scale (AIMS) score) found that the AIMS scores were not significantly influenced by HTR2A polymorphisms, despite controlling for potential confounders such as age, gender and ethnicity (Pu2009>u20090.05). Theoretically, central serotonergic function can be subject to genetic control at various other mechanistic levels including the rate of serotonin synthesis (tryptophane hydroxylase gene), release, reuptake (serotonin transporter gene) and degradation (monoamine oxidase gene). Analyses of these other serotonergic genes are indicated. In summary, polymorphisms in HTR2A do not appear to influence the risk for TD. Further studies evaluating in tandem multiple candidate genes relevant for the serotonergic system are warranted to dissect the genetic basis of the complex TD phenotype.

Excessive paternal transmission in psoriatic arthritis

OBJECTIVEnThe differential expression of a disease according to the sex of the disease-transmitting parent has been demonstrated in several autoimmune disorders. The purpose of the present study was to determine whether there are differences in the transmission and expression of psoriatic arthritis (PsA) that are dependent on the sex of the affected parent.nnnMETHODSnAll probands (patients with PsA) were identified from among the patients attending the University of Toronto Psoriatic Arthritis Clinic. A self-reported family history of psoriasis or PsA was noted for each proband. Differences in parental and offspring transmission with respect to the proband were evaluated. In addition, the expression of PsA according to the sex of the affected parent was assessed at the time of the probands presentation to the clinic.nnnRESULTSnNinety-five probands had affected parents: 62 (65%) had an affected father, and 33 (35%) had an affected mother. Thus, the proportion of paternal transmission (0.65) was significantly greater than was expected (0.5) (P = 0.001). Twelve of 74 offspring from male probands (16.2%) were affected with psoriasis or PsA, as compared with 9 of 108 offspring from female probands (8.3%) (P = 0.10). Probands whose fathers were affected had a higher frequency of skin lesions prior to arthritis (P = 0.047), an erythrocyte sedimentation rate > 15 mm/hour (P = 0.044), and a lower incidence of rheumatoid factor (P = 0.044). No differences were noted with respect to age at the onset of psoriasis or PsA, the severity of the PsA, or the frequency of HLA antigens.nnnCONCLUSIONnThere appears to be excessive paternal transmission in PsA. Further clinical confirmation and elucidation of its genetic basis is warranted.

Psychiatric epigenetics: a new focus for the new century

Among a multitude of new developments, a symposium dedicated specifically to epigenetic developments in psychiatric and related research was organized at the 7th World Congress of Psychiatric Genetics. The scientific term ‘epigenetics’ originates from epigenesis, which was one of the first developmental theories in embryology. 1 According to epigenesis, complex organisms develop from undifferentiated cells, while the competing theory of preformation stated that embryos preexist in a smaller scale in the germ cells of animal and plants. 2 While the last half of the 20th century has brought the realization that genes determine development, epigenetics has become a more adequate term to describe modifications of the genome that account for a diversity of cellular phenotypes in cells carrying identical DNA sequences. A variety of epigenetic phenomena have been identified over the last few decades. 3 Significant progress was made in understanding the epigenetic mechanisms of de novo and maintenance DNA methylation, nuclear compartmentalization, and chromatin conformation. 4

An Association Study between Schizophrenia and the Dopamine Receptor Genes DRD3 and DRD4 Using Haplotype Relative Risk

Our work investigates the relationship between genetic factors and schizophrenia, seeking to identify a gene or genes associated with the clinical form of the disease in a group of Italian patients. In pursuit of the dopaminergic hypothesis of schizophrenia, we explored a possible etiologic role of two dopamine receptor genes, DRD3 and DRD4, that have been repeatedly suggested as factors in the pathophysiology of the disease. We typed DNA polymorphisms in each of the genes that code for variation in the amino acid sequence of the receptor protein. An innovative design using parental chromosomes as controls--the haplotype relative risk strategy--represents a significant improvement over previous association studies in psychiatric genetics. Our results suggest that, at least in our well-defined population, the candidate genes DRD3 and DRD4 do not appear to play a major role in the genetic etiology of schizophrenia.

Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions.

The development and use of high throughput technologies for detailed mapping of methylated cytosines (metC) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced ‘quantitative’ C/T polymorphism that corresponds to metC/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of metC.

Molecular genetic studies of schizophrenia: challenges and insights.

Schizophrenia (SCZ) is a mental disease that affects approximately 1% of the population with life-long devastating consequences. Based on evidence for a major contribution of genetic factors, a decade of extensive efforts has been dedicated to the search of DNA sequence variations that increase the risk to SCZ. Search for genes in rare multiplex SCZ families with a large number of affected individuals and quasi-Mendelian mode of inheritance using genetic linkage methodology has been one of the favorite strategies in psychiatric genetics. Although several genomic regions were suggested for linkage to SCZ, not a single gene causing or predisposing to SCZ has been identified thus far. Furthermore, it is not clear whether the genes of familial SCZ are also involved in sporadic cases that represent the overwhelming majority of SCZ patients. For sporadic cases, genetic association studies comparing the distribution of allelic frequencies of candidate genes in SCZ patients and controls have been performed but the outcome of such studies has also been quite modest. Several factors such as possible involvement of numerous interactive genes of minor effect, yet unknown environmental effects and diagnostic ambiguities of the disease have made genetic studies in SCZ quite unproductive. In terms of future studies, a genome-wide association search is a promising approach; however, this approach requires genotyping of thousands of genetic markers in large samples. In addition, a detailed analysis of the genes, expression of which changes under the influence of environmental factors, can indicate good candidates for genetic association studies. In this connection, investigations of the epigenetic regulation of genes and not only the DNA sequence variation, may be necessary for complete understanding of the etiopathogenic mechanisms of SCZ.

Analysis of genome-wide CAG/CTG repeats, and at SEF2-1B and ERDA1 in schizophrenia and bipolar affective disorder.

A shift towards larger CAG/CTG triplet repeats and schizophrenia (SCZ) and bipolar affective disorder (BPAD) has been detected by several recent studies, using the Repeat Expansion Detection (RED) technique, however no specific loci have been shown to be responsible for this shift. Further analyses by our group of RED (CTG)10 ligation products amongst an extended sample of patients and comparison with controls matched for age, sex and ethnicity show no significant differences in distribution (Pu2009=u20090.23, nu2009=u200995; Pu2009=u20090.93, nu2009=u200991, for SCZ and BPAD respectively). Alleles at two recently discovered unstable trinucleotide repeat loci at 18q21.1 (SEF2-1B) and 17q21.3 (ERDA1) have also been analysed in affecteds and matched controls. We observed no increase in frequency of larger alleles (>37 repeats) in affected individuals at SEF2-1B (BPAD: Pu2009=u20090.95, nu2009=u2009100; SCZ: Pu2009=u20090.61, nu2009=u200997) or at ERDA1 (BPAD: Pu2009=u20090.4, nu2009=u2009101; SCZ: Pu2009=u20090.05, nu2009=u2009151, with larger alleles more frequent in controls). Our findings suggest that larger CAG/CTG repeats at these loci are neither major contributory factors to the etiology of psychosis, nor in linkage disequilibrium with a gene that is. Furthermore, when the RED results were compared to allele sizes at SEF2-1B and ERDA1, it was observed that a majority of SCZ, BPAD and control individuals with large RED products had a large allele at either or both sites (78% for RED products ⩾270 bp; 62% for RED products ⩾180 bp).