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Epigenetic gene-regulation abnormalities have been implicated in various neuropsychiatric disorders including schizophrenia and depression, as well as in the regulation of mood and anxiety. In addition, epigenetic mechanisms are involved in the actions of psychiatric drugs. Current anxiolytic drugs have significant shortcomings, and development of new medications is warranted. Two proteins, G9a (also known as EHMT2 or KMT1C) and GLP (G9a-like protein, also known as EHMT1 or KMT1D), which methylate lysine 9 of histone H3 (H3K9), could be promising anxiolytic targets. Postnatal genetic knock-out of G9a reduces anxiety-related behavior, consistent with the reduction of G9a levels by some medications used to treat anxiety (amitriptyline, imipramine and paroxetine). Conversely, there is increased anxiety-like behavior in mice with GLP haplodeficiency. We sought to determine whether two pharmacological inhibitors of G9a/GLP, UNC0642 and A-366, would have similar effects to genetic G9a/GLP insufficiency. We found that G9a/GLP inhibition with either compound reduced anxiety-like behaviors when administered to adult mice, in conjunction with decreased H3K9 methylation in the brain. In contrast, exposure to these compounds from embryonic day 9.5 (E9.5) until birth increased anxiety-like behaviors and decreased social interaction in adulthood, while H3K9 methylation was at normal levels in the brains of the adult mice. These findings reinforce genetic evidence that G9a/GLP has different effects on anxiety-like behavior at different stages of brain development, and suggest that targeting this histone methyltransferase pathway could be useful for developing new anxiolytic drugs. These data also suggest that antidepressant exposure in utero could have negative effects in adulthood, and further investigation of these effects is warranted.

The inability to digest lactose, due to lactase nonpersistence, is a common trait in adult mammals, except in certain human populations that exhibit lactase persistence. It is not known how the lactase gene is dramatically downregulated with age in most individuals but remains active in some individuals. We performed a comprehensive epigenetic study of human and mouse small intestines, by using chromosome-wide DNA-modification profiling and targeted bisulfite sequencing. Epigenetically controlled regulatory elements accounted for the differences in lactase mRNA levels among individuals, intestinal cell types and species. We confirmed the importance of these regulatory elements in modulating lactase mRNA levels by using CRISPR-Cas9-induced deletions. Genetic factors contribute to epigenetic changes occurring with age at the regulatory elements, because lactase-persistence and lactase-nonpersistence DNA haplotypes demonstrated markedly different epigenetic aging. Thus, genetic factors enable a gradual accumulation of epigenetic changes with age, thereby influencing phenotypic outcome.

Numerous recent studies have suggested that phenotypic effects of DNA sequence variants can be mediated or modulated by their epigenetic marks, such as allele-skewed DNA modification (ASM). Using Affymetrix SNP microarrays, we performed a comprehensive search of ASM effects in human post-mortem brain and sperm samples (total n = 256) from individuals with major psychosis and control individuals. Depending on the phenotypic category of the brain samples, 1.4%–7.5% of interrogated SNPs exhibited ASM effects. Next, we investigated ASM in the context of genetic studies of schizophrenia and detected that brain ASM SNPs were significantly overrepresented among sub-threshold SNPs from a schizophrenia genome-wide association study (GWAS). Brain ASM SNPs showed a much stronger enrichment in a schizophrenia GWAS than in 17 large GWASs of non-psychiatric diseases and traits, arguing that ASM effects are at least partially tissue specific. Studies of germline and control brain ASM SNPs supported a causal association between ASM and schizophrenia. Finally, significantly higher proportions of ASM SNPs than of non-ASM SNPs were detected at loci exhibiting epigenetic signatures of enhancers and promoters, and they were overrepresented within transcription factor binding regions and DNase I hypersensitive sites. All of these findings collectively indicate that ASM SNPs should be prioritized in follow-up GWASs.

Impaired neuronal processes, including dopamine imbalance, are central to the pathogenesis of major psychosis, but the molecular origins are unclear. Here we perform a multi-omics study of neurons isolated from the prefrontal cortex in schizophrenia and bipolar disorder (n = 55 cases and 27 controls). DNA methylation, transcriptomic, and genetic-epigenetic interactions in major psychosis converged on pathways of neurodevelopment, synaptic activity, and immune functions. We observe prominent hypomethylation of an enhancer within the insulin-like growth factor 2 (IGF2) gene in major psychosis neurons. Chromatin conformation analysis revealed that this enhancer targets the nearby tyrosine hydroxylase (TH) gene responsible for dopamine synthesis. In patients, we find hypomethylation of the IGF2 enhancer is associated with increased TH protein levels. In mice, Igf2 enhancer deletion disrupts the levels of TH protein and striatal dopamine, and induces transcriptional and proteomic abnormalities affecting neuronal structure and signaling. Our data suggests that epigenetic activation of the enhancer at IGF2 may enhance dopamine synthesis associated with major psychosis., Dopamine dysregulation is centrally linked to major psychosis. Here, the authors characterise the hypomethylation of an enhancer within the insulin-like growth factor 2 gene in neurons of patients with major psychosis and provide evidence that this enhancer targets the tyrosine hydroxylase gene, responsible for dopamine synthesis.

Dopamine dysregulation is central to the pathogenesis of diseases with major psychosis, but its molecular origins are unclear. In an epigenome-wide investigation in neurons, individuals with schizophrenia and bipolar disorder showed reduced DNA modifications at an enhancer in IGF2, which disrupted the regulation of the dopamine synthesis enzyme tyrosine hydroxylase and striatal dopamine levels in transgenic mice. Epigenetic control of this enhancer may be an important molecular determinant of psychosis.

New epigenetic technologies may uncover etiopathogenic mechanisms of major psychosis. In this study, we applied padlock probe-based ultra-deep bisulfite sequencing for fine mapping of modified cytosines of the HLA complex group 9 (nonprotein coding) gene in the postmortem brains of individuals affected with schizophrenia or bipolar disorder and unaffected controls. Significant differences between patients and controls were detected in both CpG and CpH modifications. In addition, we identified epigenetic age effects, DNA modification differences between sense and anti-sense strands, and demonstrated how DNA modification data can be used in clustering of patient populations. Our findings revealed new epigenetic complexities but also highlighted the potential of DNA modification approaches in the search of heterogeneous causes of major psychiatric disease.

Amyotrophic lateral sclerosis (ALS) is characterised by a loss of motor neurons, leading to paralysis. Several autosomal dominant genes were implicated in ALS pathogenesis, such as SOD1 , with over 180 reported mutations (http://alsod.iop.kcl.ac.uk/), including a p.T137A substitution.1 ,2 Recently, ARHGEF28 , encoding an RNA binding protein involved in the aggregation of light neurofilaments in ALS, was suggested as a novel ALS gene (a heterozygous K280M>fs40X mutation was detected in three patients).3 ,4 Risk of ALS may be modulated by environmental factors, sex and ageing, which could be linked to epigenetic events (eg, DNA methylation). Monozygotic (MZ) twins provide the best opportunity to investigate environmental/epigenetic factors in disease development.5 Hence, we studied ALS-discordant MZ twins, including the evaluation of DNA methylation (DNAm) age, which is an accurate predictor of chronological age across different tissues.6 It is possible that DNAm age better reflects biological age than chronological age does, since age acceleration (DNAm age minus chronological age) was associated with several disorders and mortality. Participants were recruited from the ALS Clinic at the Sunnybrook Health Sciences Centre. Genetic and epigenetic analyses were conducted as described in the online supplementary methods, including mutation analysis of SOD1 and C9orf72 , NeuroX array, reverse transcription PCR (RT-PCR), whole genome DNA methylation array (HumanMethylation450 BeadChip) and bisulfite pyrosequencing. ### Supplementary data [jnnp-2016-313592supp.pdf] The MZ twins from a Canadian PED24 family of Italian origin were 52 years old at last examination and ALS-discordant for 17 years, with the second-born twin (9377) diagnosed with ALS at 35 years (figure 1 …

Background Quantitative real-time PCR (qPCR) is the “gold-standard” technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. Results We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw Cq values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔCq method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized. Conclusions We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets.

Transcriptional variation in histologically- and genetically- identical cells is a widespread phenomenon in tissues, yet the processes conferring this heterogeneity are not well understood. To identify contributing factors, we analyzed epigenetic profiles associated with the in vivo transcriptional gradient of the mouse lactase gene (Lct), which occurs in enterocytes along the proximal-to-distal axis of the small intestine. We found that epigenetic signatures at enhancer and promoter elements aligns with transcriptional variation of Lct in enterocytes. Age and phenotype-specific environmental cues (lactose exposure after weaning) induced changes to epigenetic modifications and CTCF binding at select regulatory elements, which corresponded to the alterations in the intestinal Lct mRNA gradient. Thus, epigenetic modifications in combination with CTCF binding at regulatory elements account for the transcriptional gradient in Lct in cells of the same type. Epigenetic divergence within enterocytes may contribute to the functional specialization of intestinal subregions.

Erratum : Nat. Chem. Biol. 2011 7(9):648 2011 7(8): following 574 ; 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.

Psychiatric diseases place a tremendous burden on affected individuals, their caregivers, and the health care system. Although evidence exists for a strong inherited component to many of these conditions, dedicated efforts to identify DNA sequence-based causes have not been exceptionally productive, and very few pharmacologic treatment options are clinically available. Many features of psychiatric diseases are consistent with an epigenetic dysregulation, such as discordance of monozygotic twins, late age of onset, parent-of-origin and sex effects, and fluctuating disease course. In recent years, experimental technologies have significantly advanced, permitting in-depth studies of the epigenome and its role in maintenance of normal genomic functions, as well as disease etiopathogenesis. Here, we present an epigenetic explanation for many characteristics of psychiatric disease, review the current literature on the epigenetic mechanisms involved in major psychosis, Alzheimer's disease, and autism spectrum disorders, and describe some future directions in the field of psychiatric epigenomics.

Traditionally, etiological research of schizophrenia has been focused on elucidating predisposing genes and environmental risk factors. While numerous putative environmental hazards have been suggested, inconsistencies and methodological limitations of epidemiological studies have made it difficult to identify even a single exogenous cause of schizophrenia. Furthermore, there is increasing evidence that environmental risk factors may not play as much of a significant role in schizophrenia as previously suspected. In this article, we argue that molecular epigenetic studies can overcome the complexities of traditional epidemiological studies and may become a productive line of research in understanding the nongenetic mechanisms of schizophrenia.

Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder.

Epigenetics is a new development in complex non-Mendelian disease, which may not only uncover etiologic and pathogenic mechanisms but may also provide the basis for the development of medications that would target the primary epigenetic causes of such diseases. Such epigenetic drugs would be novel, potentially possessing substantially higher therapeutic potential and a much lower rate of adverse effects in comparison to current symptomatic treatments. A collection of epigenetic drugs already exist at various stages of development and, although their effectiveness has yet to be maximized, they show great promise in the treatment of cancer, psychiatric disorders, and other complex diseases. Here we present a review of the epigenetic theory of complex disease and an evaluation of current epigenetic therapies, as well as predictions of the future directions in this expanding field.

Twin studies have had a key role in the evaluation of heritability, a population-based estimate of the genetic contribution to phenotypic variation. These studies have led to the revelation that most normal and disease phenotypes are to some extent heritable. Recently, interest has shifted from phenomenological heritability to the identification of trait-specific genes. The era of twin studies, however, is not over: recent epigenetic and global gene expression studies suggest that the most interesting findings in twin-based research are still to come. The increasing realization of the influence of epigenetics in phenotypic outcomes means that the molecular mechanisms behind phenotypic differences in genetically identical organisms can be explored. Analyses of epigenetic twin differences and similarities might yet challenge the fundamental principles of complex biology, primarily the dogma that complex phenotypes result from DNA sequence variants interacting with the environment.

Epigenetics represents a secondary inheritance system that has been poorly investigated in human biology. The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles (P=.036 to 6.8 x 10(-14)). Second, a microarray-based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.

Gender differences in susceptibility to complex disease such as asthma, diabetes, lupus, autism and major depression, among numerous other disorders, represent one of the hallmarks of non-Mendelian biology. It has been generally accepted that endocrinological differences are involved in the sexual dimorphism of complex disease; however, specific molecular mechanisms of such hormonal effects have not been elucidated yet. This paper will review evidence that sex hormone action may be mediated via gene-specific epigenetic modifications of DNA and histones. The epigenetic modifications can explain sex effects at DNA sequence polymorphisms and haplotypes identified in gender-stratified genetic linkage and association studies. Hormone-induced DNA methylation and histone modification changes at specific gene regulatory regions may increase or reduce the risk of a disease. The epigenetic interpretation of sexual dimorphism fits well into the epigenetic theory of complex disease, which argues for the primary pathogenic role of inherited and/or acquired epigenetic misregulation rather than DNA sequence variation. The new experimental strategies, especially the high throughput microarray-based epigenetic profiling, can be used for testing the epigenetic hypothesis of gender effects in complex diseases.

Somatic DNA variation represents one of the most interesting but also one of the least investigated genetic phenomena. In addition to the classical case of DNA hypermutability at the V(D)J region, there is an increasing body of experimental evidence suggesting that genes other than immunoglobulin in tissues other than lymphocytes also exhibit nonuniformity of DNA sequence, which opens new opportunities for explaining various features of multicellular organisms. Identification of somatic DNA mutability, however, is not a trivial task and numerous confounding factors have to be taken into account. In this work we investigated putative DNA variation in the serotonin 2A receptor gene (HTR2A). A series of real-time PCR-based experiments was performed on DNA samples (n = 8) from human brain and peripheral leukocytes. Amplification of the target DNA sequences was carefully matched to that of the control plasmid containing the insert of HTR2A. Sequencing of nearly 500 clones containing a total of 150,000 nucleotides did not show any evidence for somatic DNA variation in the brain and peripheral leukocytes. It is argued in this article that although intraindividual DNA mutability may be a more common phenomenon than is generally accepted, some of the earlier claims of genetic nonidentity on the brain cells may be premature.

Despite significant effort, understanding of the molecular causes and mechanisms of bipolar disorder (BD) remains a major challenge. Numerous molecular genetic linkage and association studies have been conducted over the last two decades; however, the data are quite inconsistent or even controversial. This article develops an argument that molecular studies of BD would benefit significantly from adding an epigenetic (epiG) perspective. EpiG factors refer to modifications of DNA and chromatin that "orchestrate" the activity of the genome, including regulation of gene expression. EpiG mechanisms are consistent with various non-Mendelian features of BD such as the relatively high degree of discordance in monozygotic (MZ) twins, the critical age group for susceptibility to the disease, clinical differences in males and females, and fluctuation of the disease course, including interchanges of manic and depressive phases, among others. Apart from the phenomenological consistency, molecular epiG peculiarities may shed new light on the understanding of controversial molecular genetic findings. The relevance of epigenetics for the molecular studies of BD is demonstrated using the examples of genetic studies of BD on chromosome 11p and the X chromosome. A spectrum of epiG mechanisms such as genomic imprinting, tissue-specific effects, paramutagenesis, and epiG polymorphism, as well as epiG regulation of X chromosome inactivation, is introduced. All this serves the goal of demonstrating that epiG factors cannot be ignored anymore in complex phenotypes such as BD, and systematic large-scale epiG studies of BD have to be initiated.

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.

Elucidation of genetic factors in schizophrenia and bipolar disorder remains a challenging task to psychiatric researchers. As a rule, data from genetic linkage and association studies are quite controversial. In this article, we further explore the possibility that in addition to DNA sequence variation, a putative epigenetic dysregulation of brain genes plays an important role in the etiopathogenesis of major psychosis. We provide an epigenetic interpretation of unclear genetic findings specifically pertaining to chromosome 22 in schizophrenia and bipolar disorder. It is suggested that epigenetic strategies, when applied in conjunction with traditional genetic ones, may significantly expedite the uncovering of the molecular causes of major psychosis.

Bipolar disease (BPD) is a complex major psychiatric disorder that affects between 1% and 2% of the population and exhibits ?85% heritability. This has made BPD an appealing target for genetic studies yet, despite numerous attempts, the genetic basis of this disease remains elusive. Recently, it has come to light that epigenetic factors may also influence the development of BPD. These factors act via stable but reversible modifications of DNA and chromatin structure. In this chapter, we revisit the epidemiological, clinical, and molecular findings in BPD and reanalyze them from the perspective of inherited and acquired epigenetic misregulation. Epigenetic research has great potential to enhance our understanding of the molecular basis of BPD.

Identification of genes predisposing their carrier to complex diseases is a much more complicated task than finding genes involved in simple mendelian diseases. The slow progress in the genetic research of complex diseases could be due to limitations in the basic research strategy, which is almost exclusively orientated to the detection of disease-related DNA mutations or polymorphisms. I argue in this article that epigenetic misregulation of genes is more consistent with the features of complex diseases than is DNA sequence variation, and therefore that epigenetic factors could be important in understanding the origins of complex diseases.

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.

Genetic linkage studies of type 1 diabetes have produced a number of conflicting results, suggesting a high degree of locus heterogeneity in this disease. Approaches which model such heterogeneity will increase the power to fine map susceptibility loci. Here, using data from a genome scan of 356 affected sib pairs with type 1 diabetes, we performed heterogeneity analysis based on similarity of age at diagnosis of the sib pairs. We observed linkage to the region on chromosome 4p16.3 in sib pairs both diagnosed over the age of 10 years, whilst there was no evidence for linkage in sib pairs diagnosed before age 10 years. In contrast the sib pairs diagnosed before the age of 10 years demonstrated linkage to IDDM10, on chromosome 10p. Age of diagnosis-based heterogeneity analyses in complex diseases may be particularly helpful in mapping some susceptibility loci.

New hopes for cloning susceptibility genes for schizophrenia and bipolar affective disorder followed the discovery of a novel type of DNA mutation, namely unstable DNA. One class of unstable DNA, trinucleotide repeat expansion, is the causal mutation in myotonic dystrophy, fragile X mental retardation, Huntington disease and a number of other rare Mendelian neurological disorders. This finding has led researchers in psychiatric genetics to search for unstable DNA sites as susceptibility factors for schizophrenia and bipolar affective disorder. Increased severity and decreased age at onset of disease in successive generations, known as genetic anticipation, was reported for undifferentiated psychiatric diseases and for myotonic dystrophy early in the twentieth century, but was initially dismissed as the consequence of ascertainment bias. Because unstable DNA was demonstrated to be a molecular substrate for genetic anticipation in the majority of trinucleotide repeat diseases including myotonic dystrophy, many recent studies looking for genetic anticipation have been performed for schizophrenia and bipolar affective disorder with surprisingly consistent positive results. These studies are reviewed, with particular emphasis placed on relevant sampling and statistical considerations, and concerns are raised regarding the interpretation of such studies. In parallel, molecular genetic investigations looking for evidence of trinucleotide repeat expansion in both schizophrenia and bipolar disorder are reviewed. Initial studies of genome-wide trinucleotide repeats using the repeat expansion detection technique suggested possible association of large CAG/CTG repeat tracts with schizophrenia and bipolar affective disorder. More recently, three loci have been identified that contain large, unstable CAG/CTG repeats that occur frequently in the population and seem to account for the majority of large products identified using the repeat expansion detection method. These repeats localize to an intron in transcription factor gene SEF2-1B at 18q21, a site named ERDA1 on 17q21 with no associated coding region, and the 3' end of a gene on 13q21, SCA8, that is believed to be responsible for a form of spinocerebellar ataxia. At present no strong evidence exists that large repeat alleles at either SEF2-1B or ERDA1 are involved in the etiology of schizophrenia or bipolar disorder. Preliminary evidence suggests that large repeat alleles at SCA8 that are non-penetrant for ataxia may be a susceptibility factor for major psychosis. A fourth, but much more infrequently unstable CAG/CTG repeat has been identified within the 5' untranslated region of the gene, MAB21L1, on 13q13. A fifth CAG/CTG repeat locus has been identified within the coding region of an ion transporter, KCNN3 (hSKCa3), on 1q21. Although neither large alleles nor instability have been observed at KCNN3, this repeat locus has been extensively analyzed in association and family studies of major psychosis, with conflicting findings. Studies of polyglutamine containing genes in major psychosis have also shown some intriguing results. These findings, reviewed here, suggest that, although a major role for unstable trinucleotides in psychosis is unlikely, involvement at a more modest level in a minority of cases cannot be excluded, and warrants further investigation.

Expansion at a recently identified unstable trinucleotide repeat on chromosome 13q21 has been reported as the molecular cause for spinocerebellar ataxia type 8 (SCA8). The trinucleotide repeat, which consists of a [CTA]n repeat and adjacent [CTG]n repeat, was reported to have a pathogenic range of 107–127 CTG repeats (or 110–130 combined CTA and CTG repeats) in a large ataxia kindred. This repeat region was also cloned by our group from a bipolar affective disorder (BPAD) patient, who has approximately 600 combined repeats, and large alleles (>100 repeats) were reported to be present in 0.7% of controls and 1.5% of major psychosis patients (n = 710 and n = 1,120, respectively). We have followed up these findings by screening three new samples of BPAD and schizophrenia (SCZ) patients and controls, including 272 individuals from 14 BPAD families from Sweden, 130 individuals from 32 SCZ and BPAD families/trios from the Azores Islands, and 206 SCZ individuals from the United Kingdom and Ireland, and 219 matched controls. We found large repeat alleles above the SCA8 pathogenic range in individuals from 3 of 32 Azorean pedigrees and in 1 of 206 SCZ individuals from the United Kingdom, and repeat alleles within the SCA8 pathogenic range in 1 of 14 Swedish families. Although the rarity of major psychosis patients carrying the SCA8 expansion mutation would require a much larger sample size to reach statistical significance, these results support the previously reported observation of increased occurrence of large repeats at SCA8 in major psychosis. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:873–876, 2000. © 2000 Wiley-Liss, Inc.

A number of recent reports of linkage of markers on chromosome 10p to schizophrenia, and evidence for linkage in one study to bipolar affective disorder, provide encouragement for psychiatric genetics, after nonreplication of linkage findings at other chromosomal regions. The same region on chromosome 10 also demonstrates evidence for linkage to obesity, female alcoholism, and female type 1 diabetes. However, evidence for linkage can be confounded by the biological phenomenon of transmission ratio distortion. Transmission ratio distortion (also termed segregation distortion or meiotic drive) results in non-Mendelian segregation of alleles to live born offspring, and has not been investigated at the majority of loci for complex traits. We examined evidence for transmission ratio distortion using 40 Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees across chromosome 10 using CEPH genotype data. Evidence for linkage of females to D10S211 was found (multipoint non-parametric linkage Z score [NPL] = 1.84, P = 0.040), while there was no linkage of this marker to male sex. The observation of possible transmission ratio distortion in females on chromosome 10p requires additional study, and may impact on the interpretation of positive linkage findings in this region. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:657-661, 1999.

Several molecular and clinical similarities have been detected in Alzheimer's disease (AD) and Down syndrome (DS). The most remarkable feature is abnormal accumulation of β-amyloid in the brains of both individuals affected with AD and aging DS patients followed by dementia. In addition, AD patients exhibit dermatoglyphic patterns similar to those in DS, and late maternal age is a risk factor in both diseases. AD and DS could be related genetically because AD families exhibit a higher rate of DS cases and vice versa. Although numerous discoveries have been made in the elucidation of the etiopathogenic factors in AD and DS, little progress has been achieved in understanding the origin of the common features of the two diseases. This article reviews clinical and molecular similarities in DS and AD and also chromosome 21 studies in both diseases. A new hypothesis explaining the association between AD and DS is suggested, and this hypothesis is based on the poorly understood molecular phenomenon of aberrant meiotic recombination. Aberration in meiotic recombination has been consistently detected in chromosomal diseases including trisomy 21 and sex chromosomes. There are no studies dedicated to meiotic recombination in genetic diseases; however, evidence for disturbed recombination has been documented in several neurological diseases such as Huntington's disease, myotonic dystrophy, and fragile X syndrome. Interestingly, the rate of trisomic XXY children born to mothers transmitting fragile X mutation is higher than expected. This finding suggests that AD could be associated with DS in a similar way to which fragile X syndrome is related to trisomy of sex chromosomes. Based on analogy with fragile X syndrome, it can be predicted that AD should demonstrate aberrant meiotic recombination in chromosome 21, most likely in the region D21S1/S11–D21S16 which is linked to early onset familial AD. Based on the same rationale, different patterns of meiotic recombination in the nondisjunct chromosome 21 within DS patients grouped according to the concomitant disease are predicted.

Objective The 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. Methods All 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 proband's presentation to the clinic. Results Ninety-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. Conclusion There appears to be excessive paternal transmission in PsA. Further clinical confirmation and elucidation of its genetic basis is warranted.

A mouse model of diabetes shows gender dimorphism in the cumulative incidence of diabetes. Based on this, evidence for genetic linkage to IDDM13 on chromosome arm 2q was reported to be greater in type 1 diabetes families where there was a predominance of affected female offspring compared with families with a predominance of affected male offspring. Our objective was to investigate whether the sex of affected offspring affects evidence for linkage to susceptibility loci. Data from a genome scan of 356 affected sibpair families with type 1 diabetes were analysed to determine if there is differential evidence for linkage in families with affected children of a particular sex. At markers on chromosomes 3, 5, 7, 9, 11, and 19, we found a number of regions where the evidence for linkage is greater in families with affected sibpairs of a particular sex. Thus, evidence for linkage in families with affected sibpairs of the same gender suggests the presence of additional susceptibility loci. Several biological explanations are possible for these findings, including X and Y linkage, effects of sex hormones on gene expression, and quasi-linkage between sex chromosomes and autosomes.

Genetic anticipation is characterized by an earlier age of disease onset, increased severity, and a greater proportion of affected individuals in succeeding generations. The discovery of trinucleotide repeat expansion (TRE) mutations as the molecular correlate of anticipation in a number of rare Mendelian neurodegenerative disorders has led to a resurgence of interest in this phenomenon. Because of the difficulties presented to traditional genetics by complex diseases, the testing for genetic anticipation coupled with TRE detection has been proposed as a strategy for expediting the identification of susceptibility genes for complex disorders. In the case of breast cancer, a number of previous studies found evidence consistent with genetic anticipation. It is known that a proportion of such families are linked to either BRCA1 or BRCA2, but no TRE mutations have been identified. It has been shown that the typical ascertainment employed in studies purporting to demonstrate genetic anticipation combined with unadjusted statistical analysis can dramatically elevate the type I error. We re-examine the evidence for anticipation in breast cancer by applying a new statistical approach that appears to have validity in the analysis of anticipation to data ascertained from a recent follow-up of a large prospective cohort family study of breast cancer. Using this approach, we find no statistically significant evidence for genetic anticipation in familial breast cancer. We discuss the limitations of our analysis, including the problem of adequate sample size for this new statistical test.

DNA methylation was investigated in the putative promoter region of the human dopamine D2 receptor gene (DRD2). Twenty-two DNA samples from two types of cells differentially expressing D2 receptors, striatum and lymphocytes, were subjected to bisulphite modification-based mapping of methylated cytosines. In the tested region, the DNA from lymphocytes exhibited a significantly higher degree of methylation than that from striata. In addition, a significantly higher proportion of methylated to unmethylated cytosines was detected in DRD2 from the right than the left striatum, and a trend towards a greater degree of methylation was detected in older than in younger individuals. These DRD2 methylation findings are consistent with dopamine D2 receptor binding data from the literature which support the idea that DNA methylation plays a role in regulation of DRD2 expression.

The presence of close double recombinants in genotyping data may help identify genotyping errors. Alternatively, putative double recombinants may be associated with genetic mechanisms that may be related to disease. Phase-known apparent double recombination events were identified in the Collaborative Study on the Genetics of Alcoholism data, and compared to the sex-specific genetic map at each region. A number of double recombinants occurred within a short genetic distance. Also, in some families multiple double recombinants were observed flanking the same genetic marker, both suggesting possible genotyping error. An excess of paternal double recombinants was identified, which is consistent with reports of sex-specific differential meiotic interference.

A high degree of locus heterogeneity is likely in alcoholism, and linkage heterogeneity analysis may be helpful in mapping susceptibility loci. The genetic contribution to alcoholism in females may be higher than in males, and therefore sex of affected individuals was used in linkage analysis. Families with female alcoholics demonstrated evidence for linkage to chromosomes 10p11-p15 and 21q22.1-q22.2 while those with male alcoholics did not provide evidence for linkage to these regions. Sharing of maternal and paternal alleles was also investigated separately, and evidence for linkage of maternal alleles on chromosomes 1 and 8, and paternal alleles on chromosome 2 was observed, suggesting parental origin effects. Mapping of complex traits may benefit from tests of linkage heterogeneity based on sex, and parental origin.

The phenomenon of genomic imprinting describes the differential behavior of genes depending on their parental origin, and has been demonstrated in a few rare genetic disorders. In complex diseases, parent-of-origin effects have not been systematically studied, although there may be heuristic value in such an approach. Data from a genome scan performed using 356 affected sibling pair families with type 1 diabetes were examined looking for evidence of excess sharing of either maternal or paternal alleles. At the insulin gene (IDDM2), evidence for excess sharing of alleles transmitted from mothers was detected, which is consistent with transmission disequilibrium results published elsewhere. We also identified additional loci that demonstrate allele sharing predominantly from one parent: IDDM8 shows a paternal origin effect, IDDM10 shows a maternal effect, and a locus on chromosome 16q demonstrates a paternal effect. We have also evaluated these loci for confounding by differences in sex-specific meiotic recombination by performing linkage analysis using sex-specific genetic maps. The analysis of the parental origin of shared alleles from genome scans of complex disorders may provide additional evidence for linkage for known loci, help identify regions containing additional susceptibility loci, and assist the cloning of the genes involved.

Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.

The DRD4 dopamine receptor is thus far unique among neurotransmitter receptors in having a highly polymorphic gene structure that has been reported to produce altered receptor functioning. These allelic variations are caused by a 48-bp segment in exon III of the coding region which may be repeated from 2-10 times. Varying the numbers of repeated segments changes the length, structure, and, possibly, the functional efficiency of the receptor, which makes this gene an intriguing candidate for variations in dopamine-related behaviors, such as alcoholism and drug abuse. Thus far, these DRD4 alleles have been investigated for association with schizophrenia, bipolar disorder, Parkinson's disease, and chronic alcoholism, and all have been largely negative for a direct association. We evaluated the DRD4 genotype in 226 Finish adult males, 113 of whom were alcoholics, many of the early onset type with features of impulsivity and antisocial traits. Genotype frequencies were compared to 113 Finnish controls who were free of alcohol abuse, substance abuse, and major mental illness. In 70 alcoholics and 20 controls, we measured CSF homovanillic acid (HVA), the major metabolite of dopamine, and 5-hydroxyindoleacetic acid (5-HIAA). No association was found between a particular DRD4 dopamine receptor allele and alcoholism. CSF concentrations of the monoamine metabolites showed no significant difference among the DRD4 genotypes. This study of the DRD4 dopamine receptor in alcoholics is the first to be conducted in a clinically and ethnically homogeneous population and to relate the DRD4 genotype to CSF monoamine concentrations. The results indicate that there is no association of the DRD4 receptor with alcoholism.

Epigenetic differences are a common feature of many diseases, including cancer, and disease-associated changes have even been detected in bodily fluids. DNA modification studies in circulating DNA (cirDNA) may lead to the development of specific non-invasive biomarkers. To test this hypothesis, we investigated cirDNA modifications in prostate cancer patients with locally confined disease (n = 19), in patients with benign prostate hyperplasias (n = 20) and in men without any known prostate disease (n = 20). This initial discovery screen identified 39 disease-associated changes in cirDNA modification, and seven of these were validated using the sodium bisulfite-based mapping of modified cytosines in both the discovery cohort and an independent 38-patient validation cohort. In particular, we showed that the DNA modification of regions adjacent to the gene encoding ring finger protein 219 distinguished prostate cancer from benign hyperplasias with good sensitivity (61%) and specificity (71%). We also showed that repetitive sequences detected in this study were meaningful, as they indicated a highly statistically significant loss of DNA at the pericentromeric region of chromosome 10 in prostate cancer patients (p = 1.8 × 10(-6)). Based on these strong univariate results, we applied machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls with 72% accuracy. Lastly, we used systems biology techniques to integrate our data with publicly available DNA modification and transcriptomic data from primary prostate tumors, thereby prioritizing genes for further studies. These data suggest that cirDNA epigenomics are promising source for non-invasive biomarkers.

Understanding the origins of normal and pathological behavior is one of the most exciting opportunities in contemporary biomedical research. There is increasing evidence that, in addition to DNA sequence and the environment, epigenetic modifications of DNA and histone proteins may contribute to complex phenotypes. Inherited and/or acquired epigenetic factors are partially stable and have regulatory roles in numerous genomic activities, thus making epigenetics a promising research path in etiological studies of psychiatric disease. In this article, we review recent epigenetic studies examining the brain and other tissues, including those from individuals with schizophrenia (SCZ) and bipolar disorder (BPD). We also highlight heuristic aspects of the epigenetic theory of psychiatric disease and discuss the future directions of psychiatric epigenetics.

5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. Our goal was to characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC using glucosylation coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets and with single molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain.