Maternal depression during pregnancy and cord blood DNA methylation:Findings from the Avon Longitudinal Study of Parents and Children

Abstract Up to 13% of women may experience symptoms of depression during pregnancy or in the postpartum period. Depression during pregnancy has been associated with an increased risk of adverse neurodevelopmental outcomes in the child and epigenetic mechanisms could be one of the biological pathways to explain this association. In 844 mother–child pairs from the Avon Longitudinal Study of Parents and Children, we carried out an epigenome-wide association study (EWAS) to investigate associations between prospectively collected data on maternal depression ascertained by the Edinburgh Postnatal Depression Scale in pregnancy and DNA methylation in the cord blood of newborn offspring. In individual site analysis, we identified two CpG sites associated with maternal depression in the middle part of pregnancy. In our regional analysis, we identified 39 differentially methylated regions (DMRs). Seven DMRs were associated with depression at any time point during pregnancy, 7 associated with depression in mid-pregnancy, 23 were associated with depression in late pregnancy, and 2 DMRs were associated with depression throughout pregnancy. Several of these map to genes associated with psychiatric disease and brain development. We attempted replication in The Generation R Study and could not replicate our results. Although our findings in ALSPAC suggest that maternal depression could be associated with cord blood DNA methylation the results should be viewed as preliminary and hypothesis generating until further replicated in a larger sample

Maternal depression during pregnancy and cord blood DNA methylation: findings from the Avon Longitudinal Study of Parents and Children

Up to 13% of women may experience symptoms of depression during pregnancy or in the postpartum period. Depression during pregnancy has been associated with an increased risk of adverse neurodevelopmental outcomes in the child and epigenetic mechanisms could be one of the biological pathways to explain this association. In 844 mother–child pairs from the Avon Longitudinal Study of Parents and Children, we carried out an epigenome-wide association study (EWAS) to investigate associations between prospectively collected data on maternal depression ascertained by the Edinburgh Postnatal Depression Scale in pregnancy and DNA methylation in the cord blood of newborn offspring. In individual site analysis, we identified two CpG sites associated with maternal depression in the middle part of pregnancy. In our regional analysis, we identified 39 differentially methylated regions (DMRs). Seven DMRs were associated with depression at any time point during pregnancy, 7 associated with depression in mid-pregnancy, 23 were associated with depression in late pregnancy, and 2 DMRs were associated with depression throughout pregnancy. Several of these map to genes associated with psychiatric disease and brain development. We attempted replication in The Generation R Study and could not replicate our results. Although our findings in ALSPAC suggest that maternal depression could be associated with cord blood DNA methylation the results should be viewed as preliminary and hypothesis generating until further replicated in a larger sample

Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis

Assessment of dried blood spots for DNA methylation profiling

Background: DNA methylation reflects health-related environmental exposures and genetic risk, providing insights into aetiological mechanisms and potentially predicting disease onset, progression and treatment response. An increasingly recognised need for large-scale, longitudinally-profiled samples collected world-wide has made the development of efficient and straightforward sample collection and storage procedures a pressing issue. An alternative to the low-temperature storage of EDTA tubes of venous blood samples, which are frequently the source of the DNA used in such studies, is to collect and store at room temperature blood samples using purpose built filter paper, such as Whatman FTA® cards. Our goal was to determine whether DNA stored in this manner can be used to generate DNA methylation profiles comparable to those generated using blood samples frozen in EDTA tubes. Methods: DNA methylation profiles were obtained from matched EDTA tube and Whatman FTA® card whole-blood samples from 62 Generation Scotland: Scottish Family Health Study participants using the Infinium HumanMethylation450 BeadChip. Multiple quality control procedures were implemented, the relationship between the two sample types assessed, and epigenome-wide association studies (EWASs) performed for smoking status, age and the interaction between these variables and sample storage method. Results: Dried blood spot (DBS) DNA methylation profiles were of good quality and DNA methylation profiles from matched DBS and EDTA tube samples were highly correlated (mean r = 0.991) and could distinguish between participants. EWASs replicated established associations for smoking and age, with no evidence for moderation by storage method. Conclusions: Our results support the use of Whatman FTA® cards for collecting and storing blood samples for DNA methylation profiling. This approach is likely to be particularly beneficial for large-scale studies and those carried out in areas where freezer access is limited. Furthermore, our results will inform consideration of the use of newborn heel prick DBSs for research use

Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase

BACKGROUND: Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription. RESULTS: We demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette. CONCLUSION: Our data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile

Comparative genomic integration profiling of Sleeping Beauty transposons mobilized with high efficacy from integrase-defective lentiviral vectors in primary human cells

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer

Development of transgenic minipigs with expression of antimorphic human Cryptochrome 1

Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology,and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-a, known to be risk factors in cancer and metabolic disorders