Longitudinal analyses of the DNA methylome in deployed military servicemen identify susceptibility loci for post-traumatic stress disorder.

In order to determine the impact of the epigenetic response to traumatic stress on post-traumatic stress disorder (PTSD), this study examined longitudinal changes of genome-wide blood DNA methylation profiles in relation to the development of PTSD symptoms in two prospective military cohorts (one discovery and one replication data set). In the first cohort consisting of male Dutch military servicemen (n=93), the emergence of PTSD symptoms over a deployment period to a combat zone was significantly associated with alterations in DNA methylation levels at 17 genomic positions and 12 genomic regions. Evidence for mediation of the relation between combat trauma and PTSD symptoms by longitudinal changes in DNA methylation was observed at several positions and regions. Bioinformatic analyses of the reported associations identified significant enrichment in several pathways relevant for symptoms of PTSD. Targeted analyses of the significant findings from the discovery sample in an independent prospective cohort of male US marines (n=98) replicated the observed relation between decreases in DNA methylation levels and PTSD symptoms at genomic regions in ZFP57, RNF39 and HIST1H2APS2. Together, our study pinpoints three novel genomic regions where longitudinal decreases in DNA methylation across the period of exposure to combat trauma marks susceptibility for PTSD.

Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits.

Autism spectrum disorder (ASD) defines a group of common, complex neurodevelopmental disorders. Although the aetiology of ASD has a strong genetic component, there is considerable monozygotic (MZ) twin discordance indicating a role for non-genetic factors. Because MZ twins share an identical DNA sequence, disease-discordant MZ twin pairs provide an ideal model for examining the contribution of environmentally driven epigenetic factors in disease. We performed a genome-wide analysis of DNA methylation in a sample of 50 MZ twin pairs (100 individuals) sampled from a representative population cohort that included twins discordant and concordant for ASD, ASD-associated traits and no autistic phenotype. Within-twin and between-group analyses identified numerous differentially methylated regions associated with ASD. In addition, we report significant correlations between DNA methylation and quantitatively measured autistic trait scores across our sample cohort. This study represents the first systematic epigenomic analyses of MZ twins discordant for ASD and implicates a role for altered DNA methylation in autism.

Loss of adenylyl cyclase I activity disrupts patterning of mouse somatosensory cortex.

The somatosensory (SI) cortex of mice displays a patterned, nonuniform distribution of neurons in layer IV called the 'barrelfield' (ref. 1). Thalamocortical afferents (TCAs) that terminate in layer IV are segregated such that each barrel, a readily visible cylindrical array of neurons surrounding a cell-sparse center, represents a distinct receptive field. TCA arbors are confined to the barrel hollow and synapse on barrel-wall neurons whose dendrites are oriented toward the center of the barrel. Mice homozygous for the barrelless (brl) mutation, which occurred spontaneously in ICR stock at Université de Lausanne (Switzerland), fail to develop this patterned distribution of neurons, but still display normal topological organization of the SI cortex. Despite the absence of barrels and the overlapping zones of TCA arborization, the size of individual whisker representations, as judged by 2-deoxyglucose uptake, is similar to that of wild-type mice. We identified adenylyl cyclase type I (Adcy1) as the gene disrupted in brl mutant mice by fine mapping of proximal chromosome 11, enzyme assay, mutation analysis and examination of mice homozygous for a targeted disruption of Adcy1. These results provide the first evidence for involvement of cAMP signalling pathways in pattern formation of the brain.

Blood flow restriction training in South Africa - a panel discussion.

Background: Blood flow restriction (BFR) training uses a cuff to partially occlude venous blood flow and improve musculoskeletal training outcomes. Over the past 25 years, numerous studies have demonstrated its relative safety and efficacy. Objectives: Blood flow restriction training is under review by the Health Professions Council of South Africa due to safety and ethical concerns. The objective of this roundtable discussion is to gain better insight into the current use and perception of blood flow restriction training in South Africa. Formation of panel: The expert panel had experience with the use of BFR training and included one representative from each of the following professions, namely, sports medicine, physiotherapy and biokinetics. Discussion: The panellists provided their unique perspectives on BFR training, whilst reaching a relative consensus on its safety, screening, efficacy, and appropriate use. Agreement on appropriate loading and occlusion pressure protocols during different phases of rehabilitation was less clear. Conclusion: Although BFR is a safe and effective modality, the development of evidence-based protocols among different health professionals in South Africa is required to ensure good clinical practice.

The differential transcriptome and ontology profiles of floating and cumulus granulosa cells in stimulated human antral follicles.

Communication between various ovarian cell types is a prerequisite for folliculogenesis and ovulation. In antral follicles granulosa cells divide into two distinct populations of mural and cumulus granulosa cells (CGC), enveloping the antrum and surrounding the oocyte, respectively. Both cell types, with the mural compartment in excess, contribute to the floating granulosa cell (FGC) population in the follicular fluid. The aim of this study was to compare the transcriptomes of FGC and CGC in stimulated antral follicles obtained from 19 women undergoing IVF-ICSI procedure. FGC were obtained from follicular fluid during the follicle puncture procedure and CGC were acquired after oocyte denudation for micromanipulation. Gene expression analysis was conducted using the genome-wide Affymetrix transcriptome array. The expression profile of the two granulosa cell populations varied significantly. Out of 28 869 analysed transcripts 4480 were differentially expressed (q-value < 10(-4)) and 489 showed > or =2-fold difference in the expression level with 222 genes up-regulated in FGC and 267 in CGC. The transcriptome of FGC showed higher expression of genes involved in immune response, hematological system function and organismal injury, although CGC had genes involved in protein degradation and nervous system function up-regulated. Cell-to-cell signalling and interaction pathways were noted in both cell populations. Furthermore, numerous novel transcripts that have not been previously described in follicular physiology were identified. In conclusion, our results provide a solid basis for future studies in follicular biology that will help to identify molecular markers for oocyte and embryo viability in IVF.

Wfs1 gene deletion causes growth retardation in mice and interferes with the growth hormone pathway.

The aim of present study was to describe changes in gene expression in the temporal lobe of mice induced by deletion of the Wfs1 gene. Temporal lobes samples were analyzed using Affymetrix Mouse Genome 420 2 GeneChips and expression profiles were functionally annotated with GSEA and Ingenuity Pathway Analysis. We found that Wfs1 mutant mice are significantly smaller (20.9 +/- 1.6 g) than their wild-type counterparts (31.0 +/- 0.6 g, P < 0.0001). This difference existed in 129S6 and C57B6 backgrounds. Interestingly, microarray analysis identified upregulation of growth hormone (GH) transcripts and functional analysis revealed activation of GH pathways. In line with microarray data, the level of IGF-1 in the plasma of Wfs1 mutant mice was significantly increased (P < 0.05). Thus, Wfs1 deletion induces growth retardation, whereas the GH pathway is activated. To test the interaction between the Wfs1 deletion and genomic background, mutant mice were backcrossed to two different genetic backgrounds. In line with previous studies, an interaction between a gene knockout and genetic background was found in gene expression profiles in the congenic region. However, genetic background did not alter the effect of the Wfs1 mutation on either body weight or GH pathway activation. Further studies are needed to describe biochemical and molecular changes of the growth hormone axis as well as in other hormones to clarify their role in growth retardation in the Wfs1 mutant mice.

Quantitative trait locus association scan of early reading disability and ability using pooled DNA and 100K SNP microarrays in a sample of 5760 children.

Quantitative genetic research suggests that reading disability is the quantitative extreme of the same genetic and environmental factors responsible for normal variation in reading ability. This finding warrants a quantitative trait locus (QTL) strategy that compares low versus high extremes of the normal distribution of reading in the search for QTLs associated with variation throughout the distribution. A low reading ability group (N=755) and a high reading group (N=747) were selected from a representative UK sample of 7-year-olds assessed on two measures of reading that we have shown to be highly heritable and highly genetically correlated. The low and high reading ability groups were each divided into 10 independent DNA pools and the 20 pools were assayed on 100 K single nucleotide polymorphism (SNP) microarrays to screen for the largest allele frequency differences between the low and high reading ability groups. Seventy five of these nominated SNPs were individually genotyped in an independent sample of low (N=452) and high (N=452) reading ability children selected from a second sample of 4258 7-year-olds. Nine of the seventy-five SNPs were nominally significant (P<0.05) in the predicted direction. These 9 SNPs and 14 other SNPs showing low versus high allele frequency differences in the predicted direction were genotyped in the rest of the second sample to test the QTL hypothesis. Ten SNPs yielded nominally significant linear associations in the expected direction across the distribution of reading ability. However, none of these SNP associations accounted for more than 0.5% of the variance of reading ability, despite 99% power to detect them. We conclude that QTL effect sizes, even for highly heritable common disorders and quantitative traits such as early reading disability and ability, might be much smaller than previously considered.

Interpretation of knockout experiments: the congenic footprint.

In gene targeting experiments, the importance of genetic background is now widely appreciated, and knockout alleles are routinely backcrossed onto a standard inbred background. This produces a congenic strain with a substantial segment of embryonic stem (ES)-cell-derived chromosome still flanking the knockout allele, a phenomenon often neglected in knockout studies. In cholecystokynin 2 (Cckbr) knockout mice backcrossed with C57BL/6, we have found a clear 'congenic footprint' of expression differences in at least 10 genes across 40 Mb sequence flanking the Cckbr locus, each of which is potentially responsible for aspects of the 'knockout' phenotype. The expression differences are overwhelmingly in the knockout-low direction, which may point to a general phenomenon of background dependence. This finding emphasizes the need for caution in using gene knockouts to attribute phenotypic effects to genes. This is especially the case when the gene is of unknown function or the phenotype is unexpected, and is a particular concern for large-scale knockout and phenotypic screening programmes. However, the impact of genetic background should not be simply viewed as a potential confound, but as a unique opportunity to study the broader responses of a system to a specific (genetic) perturbation.

Building capacity for improved veterinary epidemiosurveillance in southern Africa.

A workshop to produce recommendations on training requirements for improved epidemiosurveillance of livestock diseases in southern Africa was organised at the Department of Veterinary Tropical Diseases in the Veterinary Faculty of the University of Pretoria. It was attended by 23 persons representing 10 different southern African countries. The majority of the participants were actively involved in veterinary epidemiosurveillance and many of them were members of the SADC Epidemiology and Informatics Subcommittee. Discussions focused on (i) epidemiosurveillance networks and their 2 main components, i.e. (ii) diagnosis and (iii) information flow. The debates were guided by 3 questions; (i) what are the requirements for an effective network, (ii) what cannot be achieved with existing capacity and (iii) how can the current capacity be improved. Workshop participants developed lists of realistic capacity building needs, which were divided into structural needs and training requirements. Structural needs mainly concerned communication means and quality assurance. With regard to training, the need for appropriate continuing education of all actors at the various disease management levels (non-professional, para-professional, professional) was expressed. Special emphasis was put on capacity building at the lowest level, i.e. the livestock owner and the para-professionals at the community level. At the international level, it was felt that special emphasis should be put on building capacity to improve the understanding of international agreements on trade in animals and animal products and to improve the capacity of negotiating such agreements.

Advanced paternal age effects in neurodevelopmental disorders-review of potential underlying mechanisms.

Multiple epidemiological studies suggest a relationship between advanced paternal age (APA) at conception and adverse neurodevelopmental outcomes in offspring, particularly with regard to increased risk for autism and schizophrenia. Conclusive evidence about how age-related changes in paternal gametes, or age-independent behavioral traits affect neural development is still lacking. Recent evidence suggests that the origins of APA effects are likely to be multidimensional, involving both inherited predisposition and de novo events. Here we provide a review of the epidemiological and molecular findings to date. Focusing on the latter, we present the evidence for genetic and epigenetic mechanisms underpinning the association between late fatherhood and disorder in offspring. We also discuss the limitations of the APA literature. We propose that different hypotheses relating to the origins of the APA effects are not mutually exclusive. Instead, multiple mechanisms likely contribute, reflecting the etiological complexity of neurodevelopmental disorders.

Performance deficit of alpha7 nicotinic receptor knockout mice in a delayed matching-to-place task suggests a mild impairment of working/episodic-like memory.

Patients with schizophrenia exhibit deficits in a range of cognitive functions, particularly working and episodic memory, which are thought to be core features of the disorder. Memory dysfunction in schizophrenia is familial and thus a promising endophenotype for genetic studies. Both human and animal studies suggest a role for the neural nicotinic acid receptor family in cognition and specifically the alpha7-receptor subunit in schizophrenia and its endophenotypes. Consequently, we tested mice lacking the alpha7 subunit of the neural nicotinic receptor (B6.129S7-Chrna7(tm1Bay)/J) in the delayed matching-to-place (DMP) task of the Morris water maze, a measure of working/episodic memory akin to human episodic memory. We report that a minor impairment in alpha7 knockout mice was observed in the DMP task, with knockout mice taking longer to find the hidden platform than their wildtype controls. This suggests a role for the alpha7 subunit in working/episodic memory and a potential role for the alpha7 neural nicotinic receptor gene (CHRNA7) in schizophrenia and its endophenotypes.

Interspersed repetitive sequence (IRS)-PCR for typing of whole genome radiation hybrid panels.

The typing of a radiation hybrid (RH) panel is generally achieved using a unique primer pair for each marker. We here describe a complementing approach utilizing IRS-PCR. Advantages of this technology include the use of a single universal primer to specify any locus, the rapid typing of RH lines by hybridization, and the conservative use of hybrid DNA. The technology allows the mapping of a clone without the requirement for STS generation. To test the technique, we have mapped 48 BAC clones derived from mouse chromosome 12 which we mostly identified using complex probes. As mammalian genomes are repeat-rich, the technology can easily be adapted to species other than mouse.

ViPAR: a software platform for the Virtual Pooling and Analysis of Research Data.

BACKGROUND: Research studies exploring the determinants of disease require sufficient statistical power to detect meaningful effects. Sample size is often increased through centralized pooling of disparately located datasets, though ethical, privacy and data ownership issues can often hamper this process. Methods that facilitate the sharing of research data that are sympathetic with these issues and which allow flexible and detailed statistical analyses are therefore in critical need. We have created a software platform for the Virtual Pooling and Analysis of Research data (ViPAR), which employs free and open source methods to provide researchers with a web-based platform to analyse datasets housed in disparate locations. METHODS: Database federation permits controlled access to remotely located datasets from a central location. The Secure Shell protocol allows data to be securely exchanged between devices over an insecure network. ViPAR combines these free technologies into a solution that facilitates 'virtual pooling' where data can be temporarily pooled into computer memory and made available for analysis without the need for permanent central storage. RESULTS: Within the ViPAR infrastructure, remote sites manage their own harmonized research dataset in a database hosted at their site, while a central server hosts the data federation component and a secure analysis portal. When an analysis is initiated, requested data are retrieved from each remote site and virtually pooled at the central site. The data are then analysed by statistical software and, on completion, results of the analysis are returned to the user and the virtually pooled data are removed from memory. CONCLUSIONS: ViPAR is a secure, flexible and powerful analysis platform built on open source technology that is currently in use by large international consortia, and is made publicly available at [http://bioinformatics.childhealthresearch.org.au/software/vipar/].

Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.

Techniques in mammalian genome mapping.

Increasing emphasis is being given to genomic cloning using Escherichia coli vectors of intermediate insert capacity, such as bacteriophage P1, P1-derived artificial chromosomes and the F factor based bacterial artificial chromosomes. These vectors are being used in addition to yeast artifical chromosomes (YACs) in recognition of the difficulties encountered with YAC stability and with handling of YAC DNAs (problems that will not easily be overcome). Nonetheless, YACs remain the most practical cloning system for global contig building. Efforts are currently under way to produce YAC contigs that represent the human and mouse genomes, and these will increasingly exploit extensive anchoring to detailed genetic maps. Intermediate capacity clone collections based on YAC contigs will follow, enabling the compilation of mapped gene catalogues. In this way, the era of big gene hunts will draw to a close.

Identification of genes and gene pathways associated with major depressive disorder by integrative brain analysis of rat and human prefrontal cortex transcriptomes.

Despite moderate heritability estimates, progress in uncovering the molecular substrate underpinning major depressive disorder (MDD) has been slow. In this study, we used prefrontal cortex (PFC) gene expression from a genetic rat model of MDD to inform probe set prioritization in PFC in a human post-mortem study to uncover genes and gene pathways associated with MDD. Gene expression differences between Flinders sensitive (FSL) and Flinders resistant (FRL) rat lines were statistically evaluated using the RankProd, non-parametric algorithm. Top ranking probe sets in the rat study were subsequently used to prioritize orthologous selection in a human PFC in a case-control post-mortem study on MDD from the Stanley Brain Consortium. Candidate genes in the human post-mortem study were then tested against a matched control sample using the RankProd method. A total of 1767 probe sets were differentially expressed in the PFC between FSL and FRL rat lines at (q⩽0.001). A total of 898 orthologous probe sets was found on Affymetrix's HG-U95A chip used in the human study. Correcting for the number of multiple, non-independent tests, 20 probe sets were found to be significantly dysregulated between human cases and controls at q⩽0.05. These probe sets tagged the expression profile of 18 human genes (11 upregulated and seven downregulated). Using an integrative rat-human study, a number of convergent genes that may have a role in pathogenesis of MDD were uncovered. Eighty percent of these genes were functionally associated with a key stress response signalling cascade, involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), AP-1 (activator protein 1) and ERK/MAPK, which has been systematically associated with MDD, neuroplasticity and neurogenesis.

Effects of advanced paternal age on trajectories of social behavior in offspring.

Our study is the first investigation of the effects of advanced paternal age (APA) on the developmental trajectory of social behavior in rodent offspring. Given the strong epidemiological association between APA and sexually dimorphic neurodevelopmental disorders that are characterized by abnormalities in social behavior (autism, schizophrenia), we assessed sociability in male and female inbred mice (C57BL/6J) across postnatal development (N = 104) in relation to paternal age. We found differences in early social behavior in both male and female offspring of older breeders, with differences in this social domain persisting into adulthood in males only. We showed that these social deficits were not present in the fathers of these offspring, confirming a de novo origin of an altered social trajectory in the offspring generation. Our results, highly novel in rodent research, support the epidemiological observations in humans and provide evidence for a causal link between APA, age-related changes in the paternal sperm DNA and neurodevelopmental disorders in their offspring.

Genetic and environmental (inter)actions in male mouse lines selected for aggressive and nonaggressive behavior.

The purpose of this study was to investigate the effects of genetic and environmental factors, as well as their interaction, in the etiology of aggressive behavior in two mouse lines bidirectionally selected for offensive aggression. To this end, we raised the Finnish TA (aggressive) and TNA (nonagressive) selection lines either in isolation or in cohabitation with a female after weaning. At the age of 3 months we determined their aggressive behavior in three paradigms (intruder resident, neutral cage, resident intruder) against a male standard opponent. We also determined the animals' aggressive behavior against a female mouse. The results show genetic and environmental effects, as well as gene-environment interaction. We see prominent genotype effects under all conditions but each test is sensitive to a specific combination of environmental effects. A particularly noteworthy result is that variation in the unusual behavior of aggression towards a female is largely explained by the interaction of genotype with isolation. We also examined whether test experience influenced the outcome of an encounter between an experimental animal and an opponent, and found that this factor should not be underestimated, its effect size and direction depending on the type of paradigm and way of housing. These data suggest that the identification of genes underlying aggressive behavior in mice is by no means straightforward and that the result of this search will depend on the environmental design of the study (type of paradigm, housing conditions). These data also suggest that the use of 'test battery' mice might produce different results than the use of test-naïve animals.

Home-cage activity in heterogeneous stock (HS) mice as a model of baseline activity.

Behavioral genetic work in humans indicates that clinical hyperactivity is best viewed as the extreme end of activity levels in the population. However, current animal models of hyperactivity are not studied as quantitative traits as they are either knockout models or inbred strains. Furthermore, these animal models generally demonstrate elevated locomotion in novel environments, but not in their home-cages. This is the opposite of the symptoms seen in the human condition where childhood hyperactivity is generally more pronounced in constant, unstimulating situations. In this study we filmed an outbred population of 44 heterogeneous stock (HS) mice under red light during their active phase, to assess the reliability of individual differences in home-cage behavior and extract an index of home-cage activity (HCA) level. We then compared this measure to locomotor behavior in a novel environment--the open-field. Reliable individual differences in home-cage behaviors such as running, swinging on bars, and burrowing were found, and principal component factor analysis yielded a general activity factor, which accounted for 32% of the variance and correlated 0.90 with a subjective impression of activity level. The correlation between HCA and locomotor activity in the open-field was 0.23, which was non-significant. However, the association with HCA level appeared to increase over the five minutes of the open-field, presumably as the mice habituated. Furthermore, although mice displaying particularly high and low HCA were indistinguishable early in the open-field task, they became significantly differentiated over time. We conclude that home-cage behaviors and the open-field, after habituation, display good face and construct validity, and may provide a good model of baseline activity for quantitative trait loci (QTL) discovery and functional genomics in the HS mice.