Christina A. Castellani

Ontogenetic de novo copy number variations (CNVs) as a source of genetic individuality: studies on two families with MZD twins for schizophrenia.

Genetic individuality is the foundation of personalized medicine, yet its determinants are currently poorly understood. One issue is the difference between monozygotic twins that are assumed identical and have been extensively used in genetic studies for decades [1]. Here, we report genome-wide alterations in two nuclear families each with a pair of monozygotic twins discordant for schizophrenia evaluated by the Affymetrix 6.0 human SNP array. The data analysis includes characterization of copy number variations (CNVs) and single nucleotide polymorphism (SNPs). The results have identified genomic differences between twin pairs and a set of new provisional schizophrenia genes. Samples were found to have between 35 and 65 CNVs per individual. The majority of CNVs (∼80%) represented gains. In addition, ∼10% of the CNVs were de novo (not present in parents), of these, 30% arose during parental meiosis and 70% arose during developmental mitosis. We also observed SNPs in the twins that were absent from both parents. These constituted 0.12% of all SNPs seen in the twins. In 65% of cases these SNPs arose during meiosis compared to 35% during mitosis. The developmental mitotic origin of most CNVs that may lead to MZ twin discordance may also cause tissue differences within individuals during a single pregnancy and generate a high frequency of mosaics in the population. The results argue for enduring genome-wide changes during cellular transmission, often ignored in most genetic analyses.

The effects of olanzapine on genome-wide DNA methylation in the hippocampus and cerebellum

BackgroundThe mechanism of action of olanzapine in treating schizophrenia is not clear. This research reports the effects of a therapeutic equivalent treatment of olanzapine on DNA methylation in a rat model in vivo.Genome-wide DNA methylation was assessed using a MeDIP-chip analysis. All methylated DNA immunoprecipitation (MeDIP), sample labelling, hybridization and processing were performed by Arraystar Inc (Rockville, MD, USA). The identified gene promoters showing significant alterations to DNA methylation were then subjected to Ingenuity Pathway Analysis (Ingenuity System Inc, CA, USA).ResultsThe results show that olanzapine causes an increase in methylation in 1,140, 1,294 and 1,313 genes and a decrease in methylation in 633, 565 and 532 genes in the hippocampus, cerebellum and liver, respectively. Most genes affected are tissue specific. Only 41 affected genes (approximately 3%) showed an increase and no gene showed a decrease in methylation in all three tissues. Further, the two brain regions shared 123 affected genes (approximately 10%). The affected genes are enriched in pathways affecting dopamine signalling, molecular transport, nervous system development and functions in the hippocampus; ephrin receptor signalling and synaptic long-term potentiation in the cerebellum; and tissue morphology, cellular assembly and organization in the liver. Also, the affected genes included those previously implicated in psychosis.ConclusionsThe known functions of affected genes suggest that the observed epigenetic changes may underlie the amelioration of symptoms as well as accounting for certain adverse effects including the metabolic syndrome. The results give insights into the mechanism of action of olanzapine, therapeutic effects and the side effects of antipsychotics.

Associative DNA methylation changes in children with prenatal alcohol exposure

AIM Prenatal alcohol exposure (PAE) can cause fetal alcohol spectrum disorders (FASD). Previously, we assessed PAE in brain tissue from mouse models, however whether these changes are present in humans remains unknown. MATERIALS & METHODS In this report, we show some identical changes in DNA methylation in the buccal swabs of six children with FASD using the 450K array. RESULTS The changes occur in genes related to protocadherins, glutamatergic synapses, and hippo signaling. The results were found to be similar in another heterogeneous replication group of six FASD children. CONCLUSION The replicated results suggest that children born with FASD have unique DNA methylation defects that can be influenced by sex and medication exposure. Ultimately, with future clinical development, assessment of DNA methylation from buccal swabs can provide a novel strategy for the diagnosis of FASD.

DNA methylation differences in monozygotic twin pairs discordant for schizophrenia identifies psychosis related genes and networks

BackgroundDespite their singular origin, monozygotic twin pairs often display discordance for complex disorders including schizophrenia. It is a common (1%) and often familial disease with a discordance rate of ~50% in monozygotic twins. This high discordance is often explained by the role of yet unknown environmental, random, and epigenetic factors. The involvement of DNA methylation in this disease appears logical, but remains to be established.MethodsWe have used blood DNA from two pairs of monozygotic twins discordant for schizophrenia and their parents in order to assess genome-wide methylation using a NimbleGen Methylation Promoter Microarray.ResultsThe genome-wide results show that differentially methylated regions (DMRs) exist between members representing discordant monozygotic twins. Some DMRs are shared with parent(s) and others appear to be de novo. We found twenty-seven genes affected by DMR changes that were shared in the affected member of two discordant monozygotic pairs from unrelated families. Interestingly, the genes affected by pair specific DMRs share specific networks. Specifically, this study has identified two networks; “cell death and survival” and a “cellular movement and immune cell trafficking”. These two networks and the genes affected have been previously implicated in the aetiology of schizophrenia.ConclusionsThe results are compatible with the suggestion that DNA methylation may contribute to the discordance of monozygotic twins for schizophrenia. Also, this may be accomplished by the direct effect of gene specific methylation changes on specific biological networks rather than individual genes. It supports the extensive genetic, epigenetic and phenotypic heterogeneity implicated in schizophrenia.

Copy number variation distribution in six monozygotic twin pairs discordant for schizophrenia.

We have evaluated copy number variants (CNVs) in six monozygotic twin pairs discordant for schizophrenia. The data from Affymetrix® Human SNP 6.0 arrays™ were analyzed using Affymetrix® Genotyping Console™, Partek® Genomics Suite™, PennCNV, and Golden Helix SVS™. This yielded both program-specific and overlapping results. Only CNVs called by Affymetrix Genotyping Console, Partek Genomics Suite, and PennCNV were used in further analysis. This analysis included an assessment of calls in each of the six twin pairs towards identification of unique CNVs in affected and unaffected co-twins. Real time polymerase chain reaction (PCR) experiments confirmed one CNV loss at 7q11.21 that was found in the affected patient but not in the unaffected twin. The results identified CNVs and genes that were previously implicated in mental abnormalities in four of the six twin pairs. It included PYY (twin pairs 1 and 5), EPHA3 (twin pair 3), KIAA1211L (twin pair 4), and GPR139 (twin pair 5). They represent likely candidate genes and CNVs for the discordance of four of the six monozygotic twin pairs for this heterogeneous neurodevelopmental disorder. An explanation for these differences is ontogenetic de novo events that differentiate in the monozygotic twins during development.

Autism meets schizophrenia via cadherin pathway

Search for copy number variations in a monozygotic twin pair discordant for schizophrenia has identified a deletion between two cadherin (CDH12 and CDH18) genes on 5p14. This 11Kb deletion is present in the affected but not in the unaffected twin. The results are compatible with a cadherin hypothesis for schizophrenia and complement the recent publication in Nature that implicated CDH9 and CDH10 genes of the cadherin family in autism (Wang et al 2009). Do the two diseases share cadherin as a common pathway? The identification of susceptibility factors for psychiatric disorders remains an important scientific challenge. Many of the major psychiatric disorders share overlapping clinical features including therapeutic response to specific drugs. For example, before the development of DSM III, autism was believed to be a form of childhood schizophrenia.While autism and schizophrenia have been validated as distinct disorders an increased comorbidity and overlap of symptom profile and of underlying genetic abnormalities has been noted between the two diseases (Rapoport et al., 2009). Autism starts in infancy and is characterized by deficits in communication, social interaction and by repetitive and stereotyped behavior. In contrast, schizophrenia usually manifests itself during adolescence or in early adult life. In addition to hallucinations, delusions and mood alterations, patients with schizophrenia typically have disorganized thinking, disruption of higher cognitive functions, impaired judgment and a progressive decline in social and occupational functioning. Both schizophrenia and autism have been the focus of extensive genetic studies. The results suggest that the two disorders are complex and multi-factorial and that each involve complex interaction of a number of genes and environment. Interestingly, recent hypothesis free genome-wide studies have identified genes and genetic aberrations associated with a number of psychiatric disorders including autism spectrum disorder (ASD) (Kumar and Christien, 2009) and schizophrenia (Williams et al., 2009). It has been suggested that approaches that recognize and incorporate overlapping features of such diseases may identify genes and pathways that are common in psychiatric disorders. In a recent report Wang et al. (2009) studied a cohort of 780 ASD families plus 1204 affected subjects with ASD and 6491 controls in genome-wide association (GWA). They identified six single nucleotide polymorphisms (SNPs) specific to 5p14 (particularly rs4307059) that show strong association (p=3.4× 10−8) with ASD. These SNPs are not directly associated with any gene. Rather they fall between two cadherin genes

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Olanzapine induced DNA methylation changes support the dopamine hypothesis of psychosis

BackgroundThe dopamine (DA) hypothesis of schizophrenia proposes the mental illness is caused by excessive transmission of dopamine in selected brain regions. Multiple lines of evidence, including blockage of dopamine receptors by antipsychotic drugs that are used to treat schizophrenia, support the hypothesis. However, the dopamine D2 receptor (DRD2) blockade cannot explain some important aspects of the therapeutic effect of antipsychotic drugs. In this study, we hypothesized that antipsychotic drugs could affect the transcription of genes in the DA pathway by altering their epigenetic profile.MethodsTo test this hypothesis, we examined the effect of olanzapine, a commonly used atypical antipsychotic drug, on the DNA methylation status of genes from DA neurotransmission in the brain and liver of rats. Genomic DNA isolated from hippocampus, cerebellum, and liver of olanzapine treated (n = 2) and control (n = 2) rats were analyzed using rat specific methylation arrays.ResultsOur results show that olanzapine causes methylation changes in genes encoding for DA receptors (dopamine D1 receptor, dopamine D2 receptor and dopamine D5 receptor), a DA transporter (solute carrier family 18 member 2), a DA synthesis (differential display clone 8), and a DA metabolism (catechol-O-methyltransferase). We assessed a total of 40 genes in the DA pathway and found 19 to be differentially methylated between olanzapine treated and control rats. Most (17/19) genes showed an increase in methylation, in their promoter regions with in silico analysis strongly indicating a functional potential to suppress transcription in the brain.ConclusionOur results suggest that chronic olanzapine may reduce DA activity by altering gene methylation. It may also explain the delayed therapeutic effect of antipsychotics, which occurs despite rapid dopamine blockade. Furthermore, given the common nature of epigenetic variation, this lends insight into the differential therapeutic response of psychotic patients who display adequate blockage of dopamine receptors.

Biological relevance of CNV calling methods using familial relatedness including monozygotic twins

BackgroundStudies involving the analysis of structural variation including Copy Number Variation (CNV) have recently exploded in the literature. Furthermore, CNVs have been associated with a number of complex diseases and neurodevelopmental disorders. Common methods for CNV detection use SNP, CNV, or CGH arrays, where the signal intensities of consecutive probes are used to define the number of copies associated with a given genomic region. These practices pose a number of challenges that interfere with the ability of available methods to accurately call CNVs. It has, therefore, become necessary to develop experimental protocols to test the reliability of CNV calling methods from microarray data so that researchers can properly discriminate biologically relevant data from noise.ResultsWe have developed a workflow for the integration of data from multiple CNV calling algorithms using the same array results. It uses four CNV calling programs: PennCNV (PC), Affymetrix® Genotyping Console™ (AGC), Partek® Genomics Suite™ (PGS) and Golden Helix SVS™ (GH) to analyze CEL files from the Affymetrix® Human SNP 6.0 Array™. To assess the relative suitability of each program, we used individuals of known genetic relationships. We found significant differences in CNV calls obtained by different CNV calling programs.ConclusionsAlthough the programs showed variable patterns of CNVs in the same individuals, their distribution in individuals of different degrees of genetic relatedness has allowed us to offer two suggestions. The first involves the use of multiple algorithms for the detection of the largest possible number of CNVs, and the second suggests the use of PennCNV over all other methods when the use of only one software program is desirable.

Integration of DNA sequence and DNA methylation changes in monozygotic twin pairs discordant for schizophrenia.

Schizophrenia is a complex mental disorder with high heritability (80%), extensive genetic heterogeneity, environmental contributions and only 50% concordance in discordant monozygotic (MZ) twins. Discordant MZ twins provide an exceptional opportunity to assess patient specific genome-wide genetic and epigenetic changes that may account for the disease phenotype. A combined analysis of genetic and epigenetic changes on the same twin pairs is expected to provide a more effective approach for two reasons. First, it is now possible to generate relatively reliable complete genome sequences as well as promoter methylation states on an individual level and second, the unaffected twin that originated from the same zygote provides a near perfect genetic match for contrast and comparison. This report deals with the combined analysis of DNA sequence data and methylation data on two pairs of discordant MZ twins that have been clinically followed for over 20 years. Results on Family 1 show that 58 genes differ in DNA sequence as well as promoter methylation in a schizophrenia-affected twin as compared to her healthy co-twin. The corresponding number for family 2 was 13. The two lists are over represented by neuronal genes and include a number of known schizophrenia candidate genes and drug targets. The results argue that changes in multiple genes via co-localized genetic and epigenetic alteration contribute to a liability threshold that is necessary for development of schizophrenia. This novel hypothesis, although logical, remains to be validated.