Exposure of mouse embryos to ethanol during preimplantation development: effect on DNA-methylation in the H19 imprinting control region

ABSTRACT Ethanol is a classic teratogen capable of inducing a wide range of developmental abnormalities that vary in severity, from the barely perceptible to spontaneous abortion. These defects are collectively referred to as foetal alcohol spectrum disorders (FASD). Foetal alcohol syndrome (FAS) lies at the extreme end of this spectrum and is associated with three broad domains: prenatal and/or postnatal growth retardation, distinctive facial features and brain damage. Epidemiological and animal studies clearly indicate that the clinical variability of FASD is related to four distinct window periods: preconception, preimplantation, gastrulation and postorganogenesis. These developmental windows are correlated with peak periods of epigenetic reprogramming, suggesting a common mechanism of ethanol teratogenesis. Together with experimental evidence that ethanol inhibits DNA-methyltransferase, as well as folate metabolism, this suggests an ‘epigenetic model of FASD’. The aim of the present study was to explore the validity of this model by investigating the relationship between ethanol-induced growth retardation and imprinting, following ethanol exposure during the preimplantation period. Employing an experimental study design, together with a hybrid mouse model, embryos and placentae were harvested at 10.5 days post coitus (dpc). The weights of embryos and placentae, as well as methylation profiles at the H19 imprinting control region (ICR) – an important regulator of growth - were measured. It was found that ethanol-treated embryos and placentae were severely growth retarded in comparison to controls: r=-0.760 (p<0.01, one-tailed) and r=-0.816 (p<0.05, two-tailed), respectively. Bisulphite genomic sequencing revealed that the methylation profile at the H19 ICR was unaffected in ethanol-treated embryos, in comparison to saline-treated controls. Conversely, methylation at the paternal and maternal alleles in placentae was found to be reduced and increased, respectively, in comparison to embryos. These results imply that mechanisms for the maintenance of imprinting in the embryo are more robust than in the placenta. This is consistent with the relatively longlived nature of the embryo, which must maintain imprinting for a considerably longer period of time than the placenta. Bisulphite sequencing also revealed that the paternal allele of the H19 ICR had significantly decreased levels of methylation, while the maternal allele had increased levels of methylation, in ethanol treated-placentae, in comparison to saline controls. The changes observed at the paternal allele were localized to the CTCF1 DNA-binding site, while a trend for increased methylation at the maternal allele was observed at the CTCF2 site. A partial correlation further revealed that demethylation at the paternal allele in placentae partly mediated the effect of ethanol on placental weight. An ‘epigenetic switch model’, whereby paternal Igf2 is downregulated by the epigenetic switching of the paternal allele to the maternal epigenotype, is proposed to explain this relationship. However, partial correlations also indicated that demethylation at the paternal allele of the H19 ICR, as well as placental growth retardation, did not mediate the effect of ethanol on embryo growth. Collectively, these data suggest that imprinting at the H19 ICR is not a mechanism of embryo growth retardation prior to 10.5 dpc. In explaining these results, it is proposed that the growth retarded placenta was able to meet the nutritional demands of the similarly growth retarded embryo up until 10.5 dpc. However, an important question for future research would be to examine the relationship between ethanol-induced growth retardation and imprinting during late gestation. During the final growth spurt (>14.5 dpc) the growth retarded placenta may become unable to meet the increased demands for nutrition, which would exacerbate foetal growth restriction. In sum, the present study revealed a novel mechanism of ethanol-induced growth retardation in the placenta but indicated that imprinting at the H19 ICR does not mediate the effect of ethanol on the early embryo. Further research is required to resolve the relationship between imprinting and ethanol-induced growth retardation

Exploration of a polygenic risk score for alcohol consumption:A longitudinal analysis from the ALSPAC cohort

BACKGROUND: Uncertainty remains about the true extent by which alcohol consumption causes a number of health outcomes. Genetic variants, or combinations of variants built into a polygenic risk score (PGRS), can be used in an instrumental variable framework to assess causality between a phenotype and disease outcome of interest, a method known as Mendelian randomisation (MR). We aimed to identify genetic variants involved in the aetiology of alcohol consumption, and develop a PGRS for alcohol. METHODS: Repeated measures of alcohol consumption from mothers and their offspring were collected as part of the Avon Longitudinal Study of Parents and Children. We tested the association between 89 SNPs (identified from either published GWAS data or from functional literature) and repeated measures of alcohol consumption, separately in mothers (from ages 28-48) and offspring (from ages 15-21) who had ever reported drinking. We modelled log units of alcohol using a linear mixed model and calculated beta coefficients for each SNP separately. Cross-validation was used to determine an allelic score for alcohol consumption, and the AVENGEME algorithm employed to estimate variance of the trait explained. RESULTS: Following correction for multiple testing, one SNP (rs1229984) showed evidence for association with alcohol consumption (β = -0.177, SE = 0.042, p = <0.0001) in the mothers. No SNPs showed evidence for association in the offspring after correcting for multiple testing. The optimal allelic score was generated using p-value cut offs of 0.5 and 0.05 for the mothers and offspring respectively. These scores explained 0.3% and 0.7% of the variance. CONCLUSION: Our PGRS explains a modest amount of the variance in alcohol consumption and larger sample sizes would be required to use our PGRS in an MR framework

Leucocyte telomere length and risk of cardiovascular disease: systematic review and meta-analysis.

OBJECTIVE: To assess the association between leucocyte telomere length and risk of cardiovascular disease. DESIGN: Systematic review and meta-analysis. DATA SOURCES: Studies published up to March 2014 identified through searches of Medline, Web of Science, and Embase. ELIGIBILITY CRITERIA: Prospective and retrospective studies that reported on associations between leucocyte telomere length and coronary heart disease (defined as non-fatal myocardial infarction, coronary heart disease death, or coronary revascularisation) or cerebrovascular disease (defined as non-fatal stroke or death from cerebrovascular disease) and were broadly representative of general populations--that is, they did not select cohort or control participants on the basis of pre-existing cardiovascular disease or diabetes. RESULTS: Twenty four studies involving 43,725 participants and 8400 patients with cardiovascular disease (5566 with coronary heart disease and 2834 with cerebrovascular disease) were found to be eligible. In a comparison of the shortest versus longest third of leucocyte telomere length, the pooled relative risk for coronary heart disease was 1.54 (95% confidence interval 1.30 to 1.83) in all studies, 1.40 (1.15 to 1.70) in prospective studies, and 1.80 (1.32 to 2.44) in retrospective studies. Heterogeneity between studies was moderate (I(2) = 64%, 41% to 77%, Phet<0.001) and was not significantly explained by mean age of participants (P = 0.23), the proportion of male participants (P = 0.45), or distinction between retrospective versus prospective studies (P = 0.32). Findings for coronary heart disease were similar in meta-analyses restricted to studies that adjusted for conventional vascular risk factors (relative risk 1.42, 95% confidence interval 1.17 to 1.73); studies with ≥ 200 cases (1.44, 1.20 to 1.74); studies with a high quality score (1.53, 1.22 to 1.92); and in analyses that corrected for publication bias (1.34, 1.12 to 1.60). The pooled relative risk for cerebrovascular disease was 1.42 (1.11 to 1.81), with no significant heterogeneity between studies (I(2) = 41%, 0% to 72%, Phet = 0.08). Shorter telomeres were not significantly associated with cerebrovascular disease risk in prospective studies (1.14, 0.85 to 1.54) or in studies with a high quality score (1.21, 0.83 to 1.76). CONCLUSION: Available observational data show an inverse association between leucocyte telomere length and risk of coronary heart disease independent of conventional vascular risk factors. The association with cerebrovascular disease is less certain

Best (but oft-forgotten) practices:the design, analysis, and interpretation of Mendelian randomization studies

Mendelian randomization (MR) is an increasingly important tool for appraising causality in observational epidemiology. The technique exploits the principle that genotypes are not generally susceptible to reverse causation bias and confounding, reflecting their fixed nature and Mendel’s first and second laws of inheritance. The approach is, however, subject to important limitations and assumptions that, if unaddressed or compounded by poor study design, can lead to erroneous conclusions. Nevertheless, the advent of 2-sample approaches (in which exposure and outcome are measured in separate samples) and the increasing availability of open-access data from large consortia of genome-wide association studies and population biobanks mean that the approach is likely to become routine practice in evidence synthesis and causal inference research. In this article we provide an overview of the design, analysis, and interpretation of MR studies, with a special emphasis on assumptions and limitations. We also consider different analytic strategies for strengthening causal inference. Although impossible to prove causality with any single approach, MR is a highly cost-effective strategy for prioritizing intervention targets for disease prevention and for strengthening the evidence base for public health policy

Consistent Estimation in Mendelian Randomization with Some Invalid Instruments Using a Weighted Median Estimator.

Developments in genome-wide association studies and the increasing availability of summary genetic association data have made application of Mendelian randomization relatively straightforward. However, obtaining reliable results from a Mendelian randomization investigation remains problematic, as the conventional inverse-variance weighted method only gives consistent estimates if all of the genetic variants in the analysis are valid instrumental variables. We present a novel weighted median estimator for combining data on multiple genetic variants into a single causal estimate. This estimator is consistent even when up to 50% of the information comes from invalid instrumental variables. In a simulation analysis, it is shown to have better finite-sample Type 1 error rates than the inverse-variance weighted method, and is complementary to the recently proposed MR-Egger (Mendelian randomization-Egger) regression method. In analyses of the causal effects of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol on coronary artery disease risk, the inverse-variance weighted method suggests a causal effect of both lipid fractions, whereas the weighted median and MR-Egger regression methods suggest a null effect of high-density lipoprotein cholesterol that corresponds with the experimental evidence. Both median-based and MR-Egger regression methods should be considered as sensitivity analyses for Mendelian randomization investigations with multiple genetic variants.Jack Bowden is supported by a Methodology Research Fellowship from the Medical Research Council (grant number MR/N501906/1). George Davey Smith is supported by the Medical Research Council (grant number MC UU 12013/1- 9). Philip C Haycock is supported by a Cancer Research UK Population Research Postdoctoral Fellowship. Stephen Burgess is supported by a fellowship from the Wellcome Trust (100114).This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/gepi.2196