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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.

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.

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.

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.

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.

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.

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.

We report the cloning of a gene, intronless in its coding region, which we have named APJ. This gene was cloned using the polymerase chain reaction (PCR), with a set of primers designed on the basis of the conservation that members of G protein-coupled receptors (GPCR) have in their transmembrane (TM) regions. The putative receptor protein, APJ, shares closest identity to the angiotensin receptor (AT1) ranging from 40 to 50% in the hydrophobic TM regions of these receptors. The transcripts for this gene were detected in many regions of the brain. PCR analysis of somatic cell lines found APJ-related sequences to be only present on chromosome 11, and high-resolution mapping by fluorescence in situ hybridization (FISH) sublocalized APJ on band q12.

Molecular cloning studies have now identified six HTR genes encoding the biosynthesis of the structurally homologous human serotonin (5-hydroxytryptamine; 5-HT) receptors, namely 5-HTR1A, 5-HTR1B, 5-HTR1C, 5-HTR1D, 5-HTR2 and 5-HTRS31. Several of these receptors are encoded by intronless genes, and we now report the cloning of another intronless serotonergic HTR gene. This gene was cloned by a method using the polymerase chain reaction. The nucleotide sequence of this gene is most closely homologous to the 5-HTR1D gene; however, several stop codons, frame shifts and deletions are present in the coding region suggesting that this is a pseudogene which could not encode a functional receptor. Sequence analysis also revealed that the coding sequence of this pseudogene is disrupted by insertion of a 283-bp Alu repeat sequence.