BALB/cJ is a strain susceptible to stress and extremely susceptible to a defective hedonic impact in response to chronic stressors. The strain offers much promise as an animal model for the study of stress related disorders. We present a comparative hippocampal gene expression study on the effects of unpredictable chronic mild stress on BALB/cJ and C57BL/6J mice. Affymetrix MOE 430 was used to measure hippocampal gene expression from 16 animals of two different strains (BALB/cJ and C57BL/6J) of both sexes and subjected to either unpredictable chronic mild stress (UCMS) or no stress. Differences were statistically evaluated through supervised and unsupervised linear modelling and using Weighted Gene Coexpression Network Analysis (WGCNA). In order to gain further understanding into mechanisms related to stress response, we cross-validated our results with a parallel study from the GENDEP project using WGCNA in a meta-analysis design.
The effects of UCMS are visible through Principal Component Analysis which highlights the stress sensitivity of the BALB/cJ strain. A number of genes and gene networks related to stress response were uncovered including the Creb1 gene. WGCNA and pathway analysis revealed a gene network centered on Nfkb1. Results from the meta-analysis revealed a highly significant gene pathway centred on the Ubiquitin C (Ubc) gene. All pathways uncovered are associated with inflammation and immune response.
The study investigated the molecular mechanisms underlying the response to adverse environment in an animal model using a GxE design. Stress-related differences were visible at the genomic level through PCA analysis highlighting the high sensitivity of BALB/cJ animals to environmental stressors. Several candidate genes and gene networks reported are associated with inflammation and neurogenesis and could serve to inform candidate gene selection in human studies and provide additional insight into the pathology of Major Depressive Disorder.
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The online version of this article (doi:10.1186/s12864-015-1431-6) contains supplementary material, which is available to authorized users.
BALB/cJ; UCMS; Unpredictable chronic mild stress; C57BL/6J; Stress; Ubc
The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.
Many loci in the mammalian genome are intermediately methylated. Here, by comprehensively identifying these loci and quantifying their relationship with gene activity, the authors show that intermediate methylation is an evolutionarily conserved epigenomic signature of gene regulation.
Schizophrenia is a severe neuropsychiatric disorder that is hypothesized to result from disturbances in early brain development. There is mounting evidence to support a role for developmentally regulated epigenetic variation in the molecular etiology of the disorder. Here, we describe a systematic study of schizophrenia-associated methylomic variation in the adult brain and its relationship to changes in DNA methylation across human fetal brain development.
We profile methylomic variation in matched prefrontal cortex and cerebellum brain tissue from schizophrenia patients and controls, identifying disease-associated differential DNA methylation at multiple loci, particularly in the prefrontal cortex, and confirming these differences in an independent set of adult brain samples. Our data reveal discrete modules of co-methylated loci associated with schizophrenia that are enriched for genes involved in neurodevelopmental processes and include loci implicated by genetic studies of the disorder. Methylomic data from human fetal cortex samples, spanning 23 to 184 days post-conception, indicates that schizophrenia-associated differentially methylated positions are significantly enriched for loci at which DNA methylation is dynamically altered during human fetal brain development.
Our data support the hypothesis that schizophrenia has an important early neurodevelopmental component, and suggest that epigenetic mechanisms may mediate these effects.
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Traditional diagnoses of major depressive disorder (MDD) suggested that the presence or absence of stress prior to onset results in either ‘reactive’ or ‘endogenous’ subtypes of the disorder, respectively. Several lines of research suggest that the biological underpinnings of ‘reactive’ or ‘endogenous’ subtypes may also differ, resulting in differential response to treatment. We investigated this hypothesis by comparing the gene-expression profiles of three animal models of ‘reactive’ and ‘endogenous’ depression. We then translated these findings to clinical samples using a human post-mortem mRNA study.
Affymetrix mouse whole-genome oligonucleotide arrays were used to measure gene expression from hippocampal tissues of 144 mice from the Genome-based Therapeutic Drugs for Depression (GENDEP) project. The study used four inbred mouse strains and two depressogenic ‘stress’ protocols (maternal separation and Unpredictable Chronic Mild Stress) to model ‘reactive’ depression. Stress-related mRNA differences in mouse were compared with a parallel mRNA study using Flinders Sensitive and Resistant rat lines as a model of ‘endogenous’ depression. Convergent genes differentially expressed across the animal studies were used to inform candidate gene selection in a human mRNA post-mortem case control study from the Stanley Brain Consortium.
In the mouse ‘reactive’ model, the expression of 350 genes changed in response to early stresses and 370 in response to late stresses. A minimal genetic overlap (less than 8.8%) was detected in response to both stress protocols, but 30% of these genes (21) were also differentially regulated in the ‘endogenous’ rat study. This overlap is significantly greater than expected by chance. The VAMP-2 gene, differentially expressed across the rodent studies, was also significantly altered in the human study after correcting for multiple testing.
Our results suggest that ‘endogenous’ and ‘reactive’ subtypes of depression are associated with largely distinct changes in gene-expression. However, they also suggest that the molecular signature of ‘reactive’ depression caused by early stressors differs considerably from that of ‘reactive’ depression caused by late stressors. A small set of genes was consistently dysregulated across each paradigm and in post-mortem brain tissue of depressed patients suggesting a final common pathway to the disorder. These genes included the VAMP-2 gene, which has previously been associated with Axis-I disorders including MDD, bipolar depression, schizophrenia and with antidepressant treatment response. We also discuss the implications of our findings for disease classification, personalized medicine and case-control studies of MDD.
Endogenous Depression; Reactive Depression; GENDEP; VAMP-2; DSM-IV; Stanley Brain Consortium; mRNA; Stress
Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.
Advanced paternal age is robustly associated with several human neuropsychiatric disorders, particularly autism. The precise mechanism(s) mediating the paternal age effect are not known, but they are thought to involve the accumulation of de novo (epi)genomic alterations. In this study we investigate differences in the frontal cortex transcriptome in a mouse model of advanced paternal age.
Transcriptomic profiling was undertaken for medial prefrontal cortex tissue dissected from the male offspring of young fathers (2 month old, 4 sires, n = 16 offspring) and old fathers (10 month old, 6 sires, n = 16 offspring) in a mouse model of advancing paternal age. We found a number of differentially expressed genes in the offspring of older fathers, many previously implicated in the aetiology of autism. Pathway analysis highlighted significant enrichment for changes in functional networks involved in inflammation and inflammatory disease, which are also implicated in autism.
We observed widespread alterations to the transcriptome associated with advanced paternal age with an enrichment of genes associated with inflammation, an interesting observation given previous evidence linking the immune system to several neuropsychiatric disorders including autism.
Autism; Advanced paternal age; Gene expression; Transcriptome; Inflammation; Immune response; Brain
Mood disorders consist of two etiologically related, but distinctly treated illnesses, major depressive disorder (MDD) and bipolar disorder (BPD). These disorders share similarities in their clinical presentation, and thus show high rates of misdiagnosis. Recent research has revealed significant transcriptional differences within the inflammatory cytokine pathway between MDD patients and controls, and between BPD patients and controls, suggesting this pathway may possess important biomarker properties. This exploratory study attempts to identify disorder-specific transcriptional biomarkers within the inflammatory cytokine pathway, which can distinguish between control subjects, MDD patients and BPD patients. This is achieved using RNA extracted from subject blood and applying synthesized complementary DNA to quantitative PCR arrays containing primers for 87 inflammation-related genes. Initially, we use ANOVA to test for transcriptional differences in a ‘discovery cohort’ (total n = 90) and then we use t-tests to assess the reliability of any identified transcriptional differences in a ‘validation cohort’ (total n = 35). The two most robust and reliable biomarkers identified across both the discovery and validation cohort were Chemokine (C-C motif) ligand 24 (CCL24) which was consistently transcribed higher amongst MDD patients relative to controls and BPD patients, and C-C chemokine receptor type 6 (CCR6) which was consistently more lowly transcribed amongst MDD patients relative to controls. Results detailed here provide preliminary evidence that transcriptional measures within inflammation-related genes might be useful in aiding clinical diagnostic decision-making processes. Future research should aim to replicate findings detailed in this exploratory study in a larger medication-free sample and examine whether identified biomarkers could be used prospectively to aid clinical diagnosis.
Studying the causes and correlates of natural variation in gene expression in healthy populations assumes that individual differences in gene expression can be reliably and stably assessed across time. However, this is yet to be established. We examined 4-hour test–retest reliability and 10 month test–retest stability of individual differences in gene expression in ten 12-year-old children. Blood was collected on four occasions: 10 a.m. and 2 p.m. on Day 1 and 10 months later at 10 a.m. and 2 p.m. Total RNA was hybridized to Affymetrix-U133 plus 2.0 arrays. For each probeset, the correlation across individuals between 10 a.m. and 2 p.m. on Day 1 estimates test–retest reliability. We identified 3,414 variable and abundantly expressed probesets whose 4-hour test-retest reliability exceeded .70, a conventionally accepted level of reliability, which we had 80% power to detect. Of the 3,414 reliable probesets, 1,752 were also significantly reliable 10 months later. We assessed the long-term stability of individual differences in gene expression by correlating the average expression level for each probe-set across the two 4-hour assessments on Day 1 with the average level of each probe-set across the two 4-hour assessments 10 months later. 1,291 (73.7%) of the 1,752 probe-sets that reliably detected individual differences across 4 hours on two occasions, 10 months apart, also stably detected individual differences across 10 months. Heritability, as estimated from the MZ twin intraclass correlations, is twice as high for the 1,752 reliable probesets versus all present probesets on the array (0.68 vs 0.34), and is even higher (0.76) for the 1,291 reliable probesets that are also stable across 10 months. The 1,291 probesets that reliably detect individual differences from a single peripheral blood collection and stably detect individual differences over 10 months are promising targets for research on the causes (e.g., eQTLs) and correlates (e.g., psychopathology) of individual differences in gene expression.
blood; human; individual differences; gene expression; reliability; genomewide
As the most stable and experimentally accessible epigenetic mark, DNA methylation is of great interest to the research community. The landscape of DNA methylation across tissues, through development and in disease pathogenesis is not yet well characterized. Thus there is a need for rapid and cost effective methods for assessing genome-wide levels of DNA methylation. The Illumina Infinium HumanMethylation450 (450K) BeadChip is a very useful addition to the available methods for DNA methylation analysis but its complex design, incorporating two different assay methods, requires careful consideration. Accordingly, several normalization schemes have been published. We have taken advantage of known DNA methylation patterns associated with genomic imprinting and X-chromosome inactivation (XCI), in addition to the performance of SNP genotyping assays present on the array, to derive three independent metrics which we use to test alternative schemes of correction and normalization. These metrics also have potential utility as quality scores for datasets.
The standard index of DNA methylation at any specific CpG site is β = M/(M + U + 100) where M and U are methylated and unmethylated signal intensities, respectively. Betas (βs) calculated from raw signal intensities (the default GenomeStudio behavior) perform well, but using 11 methylomic datasets we demonstrate that quantile normalization methods produce marked improvement, even in highly consistent data, by all three metrics. The commonly used procedure of normalizing betas is inferior to the separate normalization of M and U, and it is also advantageous to normalize Type I and Type II assays separately. More elaborate manipulation of quantiles proves to be counterproductive.
Careful selection of preprocessing steps can minimize variance and thus improve statistical power, especially for the detection of the small absolute DNA methylation changes likely associated with complex disease phenotypes. For the convenience of the research community we have created a user-friendly R software package called wateRmelon, downloadable from bioConductor, compatible with the existing methylumi, minfi and IMA packages, that allows others to utilize the same normalization methods and data quality tests on 450K data.
Objectives: The aim of present study was to find genetic pathways activated during infection with bacterial meningitis (BM) and potentially influencing the course of the infection using genome-wide RNA expression profiling combined with pathway analysis and functional annotation of the differential transcription.
Methods: We analyzed 21 patients with BM hospitalized in 2008. The control group consisted of 18 healthy subjects. The RNA was extracted from whole blood, globin mRNA was depleted and gene expression profiling was performed using GeneChip Human Gene 1.0 ST Arrays which can assess the transcription of 28,869 genes. Gene expression profile data were analyzed using Bioconductor packages and Bayesian modeling. Functional annotation of the enriched gene sets was used to define the altered genetic networks. We also analyzed whether gene expression profiles depend on the clinical course and outcome. In order to verify the microarray results, the expression levels of ten functionally relevant genes with high statistical significance (CD177, IL1R2, IL18R1, IL18RAP, OLFM4, TLR5, CPA3, FCER1A, IL5RA, and IL7R) were confirmed by quantitative real-time (qRT) PCR.
Results: There were 8569 genes displaying differential expression at a significance level of p < 0.05. Following False Discovery Rate (FDR) correction, a total of 5500 genes remained significant at a p-value of < 0.01. Quantitative RT-PCR confirmed the differential expression in 10 selected genes. Functional annotation and network analysis indicated that most of the genes were related to activation of humoral and cellular immune responses (enrichment score 43). Those changes were found in both adults and in children with BM compared to the healthy controls. The gene expression profiles did not significantly depend on the clinical outcome, but there was a strong influence of the specific type of pathogen underlying BM.
Conclusion: This study demonstrates that there is a very strong activation of immune response at the transcriptional level during BM and that the type of pathogen influences this transcriptional activation.
bacterial meningitis; gene expression profiling; gene networks
Insulin-like growth factor 2 (Igf2) is a paternally expressed imprinted gene regulating fetal growth, playing an integral role in the development of many tissues including the brain. The parent-of-origin specific expression of Igf2 is largely controlled by allele-specific DNA methylation at CTCF-binding sites in the imprinting control region (ICR), located immediately upstream of the neighboring H19 gene. Previously we reported evidence of a negative correlation between DNA methylation in this region and cerebellum weight in humans.
We quantified cerebellar DNA methylation across all four CTCF binding sites spanning the murine Igf2/H19 ICR in an outbred population of Heterogeneous Stock (HS) mice (n = 48). We observe that DNA methylation at the second and third CTCF binding sites in the Igf2/H19 ICR shows a negative relationship with cerebellar mass, reflecting the association observed in human post-mortem cerebellum tissue.
Given the important role of the cerebellum in motor control and cognition, and the link between structural cerebellar abnormalities and neuropsychiatric phenotypes, the identification of epigenetic factors associated with cerebellum growth and development may provide important insights about the etiology of psychiatric disorders.
Igf2; H19; Epigenetics; DNA methylation; Cerebellum; Brain; Mouse; Genotype; Genomic imprinting
miRNAs are short single-stranded non-coding RNAs involved in post-transcriptional gene regulation that play a major role in normal biological functions and diseases. Little is currently known about how expression of miRNAs is regulated. We surveyed variation in miRNA abundance in the hippocampus of mouse inbred strains, allowing us to take a genetic approach to the study of miRNA regulation, which is novel for miRNAs. The BXD recombinant inbred panel is a very well characterized genetic reference panel which allows quantitative trait locus (QTL) analysis of miRNA abundance and detection of correlates in a large store of brain and behavioural phenotypes.
We found five suggestive trans QTLs for the regulation of miRNAs investigated. Further analysis of these QTLs revealed two genes, Tnik and Phf17, under the miR-212 regulatory QTLs, whose expression levels were significantly correlated with miR-212 expression. We found that miR-212 expression is correlated with cocaine-related behaviour, consistent with a reported role for this miRNA in the control of cocaine consumption. miR-31 is correlated with anxiety and alcohol related behaviours. KEGG pathway analysis of each miRNA’s expression correlates revealed enrichment of pathways including MAP kinase, cancer, long-term potentiation, axonal guidance and WNT signalling.
The BXD reference panel allowed us to establish genetic regulation and characterize biological function of specific miRNAs. QTL analysis enabled detection of genetic loci that regulate the expression of these miRNAs. eQTLs that regulate miRNA abundance are a new mechanism by which genetic variation influences brain and behaviour. Analysis of one of these QTLs revealed a gene, Tnik, which may regulate the expression of a miRNA, a molecular pathway and a behavioural phenotype. Evidence of genetic covariation of miR-212 abundance and cocaine related behaviours is strongly supported by previous functional studies, demonstrating the value of this approach for discovery of new functional roles and downstream processes regulated by miRNA.
Disruption of the circadian rhythm is a key feature of bipolar disorder. Variation in genes encoding components of the molecular circadian clock has been associated with increased risk of the disorder in clinical populations. Similarly in animal models, disruption of the circadian clock can result in altered mood and anxiety which resemble features of human mania; including hyperactivity, reduced anxiety and reduced depression-like behaviour. One such mutant, after hours (Afh), an ENU-derived mutant with a mutation in a recently identified circadian clock gene Fbxl3, results in a disturbed (long) circadian rhythm of approximately 27 hours.
Anxiety, exploratory and depression-like behaviours were evaluated in Afh mice using the open-field, elevated plus maze, light-dark box, holeboard and forced swim test. To further validate findings for human mania, polymorphisms in the human homologue of FBXL3, genotyped by three genome wide case control studies, were tested for association with bipolar disorder.
Afh mice showed reduced anxiety- and depression-like behaviour in all of the behavioural tests employed, and some evidence of increased locomotor activity in some tests. An analysis of three separate human data sets revealed a gene wide association between variation in FBXL3 and bipolar disorder (P = 0.009).
Our results are consistent with previous studies of mutants with extended circadian periods and suggest that disruption of FBXL3 is associated with mania-like behaviours in both mice and humans.
Dynamic changes to the epigenome play a critical role in establishing and maintaining cellular phenotype during differentiation, but little is known about the normal methylomic differences that occur between functionally distinct areas of the brain. We characterized intra- and inter-individual methylomic variation across whole blood and multiple regions of the brain from multiple donors.
Distinct tissue-specific patterns of DNA methylation were identified, with a highly significant over-representation of tissue-specific differentially methylated regions (TS-DMRs) observed at intragenic CpG islands and low CG density promoters. A large proportion of TS-DMRs were located near genes that are differentially expressed across brain regions. TS-DMRs were significantly enriched near genes involved in functional pathways related to neurodevelopment and neuronal differentiation, including BDNF, BMP4, CACNA1A, CACA1AF, EOMES, NGFR, NUMBL, PCDH9, SLIT1, SLITRK1 and SHANK3. Although between-tissue variation in DNA methylation was found to greatly exceed between-individual differences within any one tissue, we found that some inter-individual variation was reflected across brain and blood, indicating that peripheral tissues may have some utility in epidemiological studies of complex neurobiological phenotypes.
This study reinforces the importance of DNA methylation in regulating cellular phenotype across tissues, and highlights genomic patterns of epigenetic variation across functionally distinct regions of the brain, providing a resource for the epigenetics and neuroscience research communities.
Gene Set Enrichment (GSE) is a computational technique which determines whether a priori defined set of genes show statistically significant differential expression between two phenotypes. Currently, the gene sets used for GSE are derived from annotation or pathway databases, which often contain computationally based and unrepresentative data. Here, we propose a novel approach for the generation of comprehensive and biologically derived gene sets, deriving sets through the application of machine learning techniques to gene expression data. These gene sets can be produced for specific tissues, developmental stages or environments. They provide a powerful and functionally meaningful way in which to mine genomewide association and next generation sequencing data in order to identify disease-associated variants and pathways.
gene set enrichment; annotation database; gene expression data; machine learning; next generation sequencing
Studies of the major psychoses, schizophrenia (SZ) and bipolar disorder (BD), have traditionally focused on genetic and environmental risk factors, although more recent work has highlighted an additional role for epigenetic processes in mediating susceptibility. Since monozygotic (MZ) twins share a common DNA sequence, their study represents an ideal design for investigating the contribution of epigenetic factors to disease etiology. We performed a genome-wide analysis of DNA methylation on peripheral blood DNA samples obtained from a unique sample of MZ twin pairs discordant for major psychosis. Numerous loci demonstrated disease-associated DNA methylation differences between twins discordant for SZ and BD individually, and together as a combined major psychosis group. Pathway analysis of our top loci highlighted a significant enrichment of epigenetic changes in biological networks and pathways directly relevant to psychiatric disorder and neurodevelopment. The top psychosis-associated, differentially methylated region, significantly hypomethylated in affected twins, was located in the promoter of ST6GALNAC1 overlapping a previously reported rare genomic duplication observed in SZ. The mean DNA methylation difference at this locus was 6%, but there was considerable heterogeneity between families, with some twin pairs showing a 20% difference in methylation. We subsequently assessed this region in an independent sample of postmortem brain tissue from affected individuals and controls, finding marked hypomethylation (>25%) in a subset of psychosis patients. Overall, our data provide further evidence to support a role for DNA methylation differences in mediating phenotypic differences between MZ twins and in the etiology of both SZ and BD.
Methcathinone (ephedrone) is relatively easily accessible for abuse. Its users develop an extrapyramidal syndrome and it is not known if this is caused by methcathinone itself, by side-ingredients (manganese), or both. In the present study we aimed to clarify molecular mechanisms underlying this condition. We used microarrays to analyze whole-genome gene expression patterns of peripheral blood from 20 methcathinone users and 20 matched controls. Gene expression profile data were analyzed by Bayesian modeling and functional annotation. Of 28,869 genes on the microarrays, 326 showed statistically significant differential expression with FDR adjusted p-values below 0.05. Quantitative real-time PCR confirmed differential expression for the most of the genes selected for validation. Functional annotation and network analysis indicated activation of a gene network that included immunological disease, cellular movement, and cardiovascular disease functions (enrichment score 42). As HIV and HCV infections were confounding factors, we performed additional stratification of subjects. A similar functional activation of the “immunological disease” category was evident when we compared subjects according to injection status (past versus current users, balanced for HIV and HCV infection). However, this difference was not large therefore the major effect was related to the HIV status of the subjects. Mn–methcathinone abusers have blood RNA expression patterns that mostly reflect their HIV and HCV infections.
ephedrone; gene expression profiling; HIV; intravenous drug abuse; methcathinone; manganese
We report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism) microarrays and DNA pooling (SNP-MaP) employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations). The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes.
In the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods.
Despite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.
Across the genome, outside of a small number of known imprinted genes and regions subject to X-inactivation in females, DNA methylation at CpG dinucleotides is often assumed to be complementary across both alleles in a diploid cell. However, recent findings suggest the reality is more complex, with the discovery that allele-specific methylation (ASM) is a common feature across the human genome. A key observation is that the majority of ASM is associated with genetic variation in cis, although a noticeable proportion is also non-cis in nature and mediated, for example, by parental origin. ASM appears to be both quantitative, characterized by subtle skewing of DNA methylation between alleles, and heterogeneous, varying across tissues and between individuals. These findings have important implications for complex disease genetics; while cis-mediated ASM provides a functional consequence for non-coding genetic variation, heterogeneous and quantitative ASM complicates the identification of disease-associated loci. We propose that non-cis ASM could contribute toward the “missing heritability” of complex diseases, rendering certain loci hemizygous and masking the direct association between genotype and phenotype. We suggest that the interpretation of results from genome-wide association studies can be improved by the incorporation of epi-allelic information and that in order to fully understand the extent and consequence of ASM in the human genome, a comprehensive sequencing-based analysis of allelic methylation patterns across tissues and individuals is required.
DNA methylation; allelespecific methylation; allele-specific expression; tissue-specific methylation; epigenetics; imprinting; genome-wide association study (GWAS); genetics; complex disease; missing heritability
Childhood general cognitive ability (g) is important for a wide range of outcomes in later life, from school achievement to occupational success and life expectancy. Large-scale association studies will be essential in the quest to identify variants that make up the substantial genetic component implicated by quantitative genetic studies. We conducted a three-stage genome-wide association study for general cognitive ability using over 350,000 single nucleotide polymorphisms (SNPs) in the quantitative extremes of a population sample of 7,900 7-year-old children from the UK Twins Early Development Study. Using two DNA pooling stages to enrich true positives, each of around 1,000 children selected from the extremes of the distribution, and a third individual genotyping stage of over 3,000 children to test for quantitative associations across the normal range, we aimed to home in on genes of small effect. Genome-wide results suggested that our approach was successful in enriching true associations and 28 SNPs were taken forward to individual genotyping in an unselected population sample. However, although we found an enrichment of low P values and identified nine SNPs nominally associated with g (P < 0.05) that show interesting characteristics for follow-up, further replication will be necessary to meet rigorous standards of association. These replications may take advantage of SNP sets to overcome limitations of statistical power. Despite our large sample size and three-stage design, the genes associated with childhood g remain tantalizingly beyond our current reach, providing further evidence for the small effect sizes of individual loci. Larger samples, denser arrays and multiple replications will be necessary in the hunt for the genetic variants that influence human cognitive ability.
Electronic supplementary material
The online version of this article (doi:10.1007/s10519-010-9350-4) contains supplementary material, which is available to authorized users.
Genetics; Genome-wide association; General cognitive ability; Intelligence; Population sample; Middle childhood
Accumulating evidence from epidemiological research has demonstrated an association between advanced paternal age and risk for several psychiatric disorders including autism, schizophrenia and early-onset bipolar disorder. In order to establish causality, this study used an animal model to investigate the effects of advanced paternal age on behavioural deficits in the offspring.
C57BL/6J offspring (n = 12 per group) were bred from fathers of two different ages, 2 months (young) and 10 months (old), and mothers aged 2 months (n = 6 breeding pairs per group). Social and exploratory behaviors were examined in the offspring.
The offspring of older fathers were found to engage in significantly less social (p = 0.02) and exploratory (p = 0.02) behaviors than the offspring of younger fathers. There were no significant differences in measures of motor activity.
Given the well-controlled nature of this study, this provides the strongest evidence for deleterious effects of advancing paternal age on social and exploratory behavior. De-novo chromosomal changes and/or inherited epigenetic changes are the most plausible explanatory factors.
Two separate genome-wide association studies were conducted to identify single nucleotide polymorphisms (SNPs) associated with social and nonsocial autistic-like traits. We predicted that we would find SNPs associated with social and non-social autistic-like traits and that different SNPs would be associated with social and nonsocial. In Stage 1, each study screened for allele frequency differences in ~430,000 autosomal SNPs using pooled DNA on microarrays in high-scoring versus low-scoring boys from a general population sample (N = ~400/group). In Stage 2, 22 and 20 SNPs in the social and non-social studies, respectively, were tested for QTL association by individually genotyping an independent community sample of 1,400 boys. One SNP (rs11894053) was nominally associated (P < .05, uncorrected for multiple testing) with social autistic-like traits. When the sample was increased by adding females, 2 additional SNPs were nominally significant (P < .05). These 3 SNPs, however, showed no significant association in transmission disequilibrium analyses of diagnosed ASD families.
Autistic traits; Genome-wide association; Autism; Microarrays; Heritability; Pooling
Micro-RNAs (miRNAs) are short, single-stranded, noncoding RNAs that are involved in the regulation of protein-coding genes at the level of messenger RNA (mRNA). They are involved in the regulation of numerous traits, including developmental timing, apoptosis, immune function, and neuronal development. To better understand how the expression of the miRNAs themselves is regulated, we looked for miRNA expression differences among four mouse inbred strains, A/J, BALB/cJ, C57BL/6J, and DBA/2J, in one tissue, the hippocampus. A total of 166 miRNA RT-PCR assays were used to screen RNA pools for each strain. Twenty miRNA species that were markedly different between strains were further investigated using eight individual samples per strain, and 11 miRNAs showed significant differences across strains (p < 0.05). This is the first observation of miRNA expression differences across inbred mice strains. We conducted an in silico correlation analysis of the expression of these differentially expressed miRNAs with phenotype data and mRNA expression to better characterise the effects of these miRNAs on both phenotype and the regulation of mRNA expression. This approach has allowed us to nominate miRNAs that have potential roles in anxiety, exploration, and learning and memory.
Electronic supplementary material
The online version of this article (doi:10.1007/s00335-008-9116-y) contains supplementary material, which is available to authorized users.
Microarrays are designed to measure genome-wide differences in gene expression. In cases where a tissue is not accessible for analysis (e.g. human brain), it is of interest to determine whether a second, accessible tissue could be used as a surrogate for transcription profiling. Surrogacy has applications in the study of behavioural and neurodegenerative disorders. Comparison between hippocampus and spleen mRNA obtained from a mouse recombinant inbred panel indicates a high degree of correlation between the tissues for genes that display a high heritability of expression level. This correlation is not limited to apparent expression differences caused by sequence polymorphisms in the target sequences and includes both cis and trans genetic effects. A tissue such as blood could therefore give surrogate information on expression in brain for a subset of genes, in particular those co-expressed between the two tissues, which have heritably varying expression.
hippocampus; spleen; recombinant inbred strain; gene expression; surrogacy
Differences in gene expression in the CNS influence behavior and disease susceptibility. To systematically explore the role of normal variation in expression on hippocampal structure and function, we generated an online microarray database for a diverse panel of strains of mice, including most common inbred strains and numerous recombinant inbred lines (www.genenetwork.org). Using this resource, coexpression networks for families of genes can be generated rapidly to test causal models related to function. The data set is optimized for quantitative trait locus (QTL) mapping and was used to identify over 5500 QTLs that modulate mRNA levels. We describe a wide variety of analyses and novel synthetic approaches that take advantage of this resource, and demonstrate how both the data and associated tools can be applied to the study of gene regulation in the hippocampus and relations to structure and function.
recombinant inbred mice; hippocampus; QTL; genetical genomics; transcript expression