A systematic review of neuroimaging epigenetic research: calling for an increased focus on development.

Epigenetic mechanisms, such as DNA methylation (DNAm), have gained increasing attention as potential biomarkers and mechanisms underlying risk for neurodevelopmental, psychiatric and other brain-based disorders. Yet, surprisingly little is known about the extent to which DNAm is linked to individual differences in the brain itself, and how these associations may unfold across development - a time of life when many of these disorders emerge. Here, we systematically review evidence from the nascent field of Neuroimaging Epigenetics, combining structural or functional neuroimaging measures with DNAm, and the extent to which the developmental period (birth to adolescence) is represented in these studies. We identified 111 articles published between 2011-2021, out of which only a minority (21%) included samples under 18 years of age. Most studies were cross-sectional (85%), employed a candidate-gene approach (67%), and examined DNAm-brain associations in the context of health and behavioral outcomes (75%). Nearly half incorporated genetic data, and a fourth investigated environmental influences. Overall, studies support a link between peripheral DNAm and brain imaging measures, but there is little consistency in specific findings and it remains unclear whether DNAm markers present a cause, correlate or consequence of brain alterations. Overall, there is large heterogeneity in sample characteristics, peripheral tissue and brain outcome examined as well as the methods used. Sample sizes were generally low to moderate (median nall = 98, ndevelopmental = 80), and attempts at replication or meta-analysis were rare. Based on the strengths and weaknesses of existing studies, we propose three recommendations on how advance the field of Neuroimaging Epigenetics. We advocate for: (1) a greater focus on developmentally oriented research (i.e. pre-birth to adolescence); (2) the analysis of large, prospective, pediatric cohorts with repeated measures of DNAm and imaging to assess directionality; and (3) collaborative, interdisciplinary science to identify robust signals, triangulate findings and enhance translational potential.

Epigenome-wide change and variation in DNA methylation in childhood: trajectories from birth to late adolescence.

DNA methylation (DNAm) is known to play a pivotal role in childhood health and development, but a comprehensive characterization of genome-wide DNAm trajectories across this age period is currently lacking. We have therefore performed a series of epigenome-wide association studies in 5019 blood samples collected at multiple time-points from birth to late adolescence from 2348 participants of two large independent cohorts. DNAm profiles of autosomal CpG sites (CpGs) were generated using the Illumina Infinium HumanMethylation450 BeadChip. Change over time was widespread, observed at over one-half (53%) of CpGs. In most cases, DNAm was decreasing (36% of CpGs). Inter-individual variation in linear trajectories was similarly widespread (27% of CpGs). Evidence for non-linear change and inter-individual variation in non-linear trajectories was somewhat less common (11 and 8% of CpGs, respectively). Very little inter-individual variation in change was explained by sex differences (0.4% of CpGs) even though sex-specific DNAm was observed at 5% of CpGs. DNAm trajectories were distributed non-randomly across the genome. For example, CpGs with decreasing DNAm were enriched in gene bodies and enhancers and were annotated to genes enriched in immune-developmental functions. In contrast, CpGs with increasing DNAm were enriched in promoter regions and annotated to genes enriched in neurodevelopmental functions. These findings depict a methylome undergoing widespread and often non-linear change throughout childhood. They support a developmental role for DNA methylation that extends beyond birth into late adolescence and has implications for understanding life-long health and disease. DNAm trajectories can be visualized at http://epidelta.mrcieu.ac.uk.

A robust and standardized method to isolate and expand mesenchymal stromal cells from human umbilical cord.

BACKGROUND AIMS: Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion. METHODS: Using 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics. RESULTS: We demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of ∼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity. CONCLUSIONS: We present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies.

Pooled analysis of epigenome-wide association studies of food consumption in KORA, TwinsUK and LLS.

PURPOSE: Examining epigenetic patterns is a crucial step in identifying molecular changes of disease pathophysiology, with DNA methylation as the most accessible epigenetic measure. Diet is suggested to affect metabolism and health via epigenetic modifications. Thus, our aim was to explore the association between food consumption and DNA methylation. METHODS: Epigenome-wide association studies were conducted in three cohorts: KORA FF4, TwinsUK, and Leiden Longevity Study, and 37 dietary exposures were evaluated. Food group definition was harmonized across the three cohorts. DNA methylation was measured using Infinium MethylationEPIC BeadChip in KORA and Infinium HumanMethylation450 BeadChip in the Leiden study and the TwinsUK study. Overall, data from 2293 middle-aged men and women were included. A fixed-effects meta-analysis pooled study-specific estimates. The significance threshold was set at 0.05 for false-discovery rate-adjusted p values per food group. RESULTS: We identified significant associations between the methylation level of CpG sites and the consumption of onions and garlic (2), nuts and seeds (18), milk (1), cream (11), plant oils (4), butter (13), and alcoholic beverages (27). The signals targeted genes of metabolic health relevance, for example, GLI1, RPTOR, and DIO1, among others. CONCLUSION: This EWAS is unique with its focus on food groups that are part of a Western diet. Significant findings were mostly related to food groups with a high-fat content.

Omixer: multivariate and reproducible sample randomization to proactively counter batch effects in omics studies.

MOTIVATION: Batch effects heavily impact results in omics studies, causing bias and false positive results, but software to control them preemptively is lacking. Sample randomization prior to measurement is vital for minimizing these effects, but current approaches are often ad hoc, poorly documented and ill-equipped to handle multiple batches and outcomes. RESULTS: We developed Omixer-a Bioconductor package implementing multivariate and reproducible sample randomization for omics studies. It proactively counters correlations between technical factors and biological variables of interest by optimizing sample distribution across batches. AVAILABILITYAND IMPLEMENTATION: Omixer is available from Bioconductor at http://bioconductor.org/packages/release/bioc/html/Omixer.html. Scripts and data used to generate figures available upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Impact of placental sharing and large bidirectional anastomoses on birthweight discordance in monochorionic twins: a retrospective cohort study in 449 cases.

BACKGROUND: In monochorionic twin pregnancies, the fetuses share a single placenta. When this placenta is unequally shared, a discordant antenatal growth pattern ensues resulting in high rates of perinatal morbidity and mortality. Understanding placental pathophysiology is paramount in devising feasible antenatal management strategies. Unequal placental sharing is not the sole determinant of birthweight discordance as there is no one-to-one relationship with placental share discordance. Placental angioarchitecture, especially the presence of large bidirectional anastomoses, is thought to affect this relationship by allowing for a compensatory intertwin blood flow. OBJECTIVE: This study aimed to assess whether placental angioarchitecture can affect birthweight discordance in live-born monochorionic twins, the aim of our study was 2-fold: (1) to assess the relationship between birthweight discordance and placental share discordance and (2) to examine to what extent large bidirectional anastomoses can compensate for the effect of unequal placental sharing on birthweight discordance, with a subgroup analysis for umbilical artery Doppler flow patterns in cases with a birthweight discordance of ≥20%. STUDY DESIGN: This was a retrospective cohort study that included monochorionic twin pregnancies observed in our center between March 2002 and June 2021, in which twins with a birthweight discordance of ≥20% were classified according to umbilical artery Doppler flow patterns of the smaller twin. We excluded cases with twin-twin transfusion syndrome and twin anemia polycythemia sequence. Monochorionic placentas of live-born twins were injected with dye, and images were saved for computer measurements of placental sharing and the diameter of anastomoses. Univariate linear regressions of the relationship between placental share discordance and birthweight discordance (both calculated as larger weight or share-smaller weight or share/larger weight or share×100%) and the relationship between arterioarterial and venovenous diameters and birthweight ratio/placental territory ratio were performed. RESULTS: A total of 449 placentas were included in the analysis. Placental share discordance was positively correlated with birthweight discordance (ß coefficient, 0.325; 95% confidence interval, 0.254-0.397; P<.0001). The arterioarterial diameter was negatively correlated with birthweight ratio/placental territory ratio (ß coefficient, -0.041; 95% confidence interval, -0.059 to -0.023; P<.0001), meaning that an increase in arterioarterial diameter leads to less birthweight discordance than expected for the amount of placental share discordance. There was no relationship between venovenous diameter and birthweight ratio/placental territory ratio (ß coefficient, -0.007; 95% confidence interval, -0.027 to 0.012; P=.473). CONCLUSION: Birthweight discordance in monochorionic twins was strongly associated with placental share discordance. Large arterioarterial anastomoses can mitigate the effect of unequal placental sharing.

Investigating the relationships between unfavourable habitual sleep and metabolomic traits: evidence from multi-cohort multivariable regression and Mendelian randomization analyses.

BACKGROUND: Sleep traits are associated with cardiometabolic disease risk, with evidence from Mendelian randomization (MR) suggesting that insomnia symptoms and shorter sleep duration increase coronary artery disease risk. We combined adjusted multivariable regression (AMV) and MR analyses of phenotypes of unfavourable sleep on 113 metabolomic traits to investigate possible biochemical mechanisms linking sleep to cardiovascular disease. METHODS: We used AMV (N = 17,368) combined with two-sample MR (N = 38,618) to examine effects of self-reported insomnia symptoms, total habitual sleep duration, and chronotype on 113 metabolomic traits. The AMV analyses were conducted on data from 10 cohorts of mostly Europeans, adjusted for age, sex, and body mass index. For the MR analyses, we used summary results from published European-ancestry genome-wide association studies of self-reported sleep traits and of nuclear magnetic resonance (NMR) serum metabolites. We used the inverse-variance weighted (IVW) method and complemented this with sensitivity analyses to assess MR assumptions. RESULTS: We found consistent evidence from AMV and MR analyses for associations of usual vs. sometimes/rare/never insomnia symptoms with lower citrate (- 0.08 standard deviation (SD)[95% confidence interval (CI) - 0.12, - 0.03] in AMV and - 0.03SD [- 0.07, - 0.003] in MR), higher glycoprotein acetyls (0.08SD [95% CI 0.03, 0.12] in AMV and 0.06SD [0.03, 0.10) in MR]), lower total very large HDL particles (- 0.04SD [- 0.08, 0.00] in AMV and - 0.05SD [- 0.09, - 0.02] in MR), and lower phospholipids in very large HDL particles (- 0.04SD [- 0.08, 0.002] in AMV and - 0.05SD [- 0.08, - 0.02] in MR). Longer total sleep duration associated with higher creatinine concentrations using both methods (0.02SD per 1 h [0.01, 0.03] in AMV and 0.15SD [0.02, 0.29] in MR) and with isoleucine in MR analyses (0.22SD [0.08, 0.35]). No consistent evidence was observed for effects of chronotype on metabolomic measures. CONCLUSIONS: Whilst our results suggested that unfavourable sleep traits may not cause widespread metabolic disruption, some notable effects were observed. The evidence for possible effects of insomnia symptoms on glycoprotein acetyls and citrate and longer total sleep duration on creatinine and isoleucine might explain some of the effects, found in MR analyses of these sleep traits on coronary heart disease, which warrant further investigation.

DNA methylation differences at birth after conception through ART.

STUDY QUESTION: Is there a relation between ART and DNA methylation (DNAm) patterns in cord blood, including any differences between IVF and ICSI? SUMMARY ANSWER: DNAm at 19 CpGs was associated with conception via ART, with no difference found between IVF and ICSI. WHAT IS KNOWN ALREADY: Prior studies on either IVF or ICSI show conflicting outcomes, as both widespread effects on DNAm and highly localized associations have been reported. No study on both IVF and ICSI and genome-wide neonatal DNAm has been performed. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional study comprising 87 infants conceived with IVF or ICSI and 70 conceived following medically unassisted conception. The requirement for inclusion in the study was an understanding of the Swedish language and exclusion was the use of donor gametes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were from the UppstART study, which was recruited from fertility and reproductive health clinics, and the Born into Life cohort, which is recruited from the larger LifeGene study. We measured DNAm from DNA extracted from cord blood collected at birth using a micro-array (450k array). Group differences in DNAm at individual CpG dinucleotides (CpGs) were determined using robust linear models and post-hoc Tukey's tests. MAIN RESULTS AND THE ROLE OF CHANCE: We found no association of ART conception with global methylation levels, imprinted loci and meta-stable epialleles. In contrast, we identify 19 CpGs at which DNAm was associated with being conceived via ART (effect estimates: 0.5-4.9%, PFDR < 0.05), but no difference was found between IVF and ICSI. The associated CpGs map to genes related to brain function/development or genes connected to the plethora of conditions linked to subfertility, but functional annotation did not point to any likely functional consequences. LIMITATIONS, REASONS FOR CAUTION: We measured DNAm in cord blood and not at later ages or in other tissues. Given the number of tests performed, our study power is limited and the findings need to be replicated in an independent study. WIDER IMPLICATIONS OF THE FINDINGS: We find that ART is associated with DNAm differences in cord blood when compared to non-ART samples, but these differences are limited in number and effect size and have unknown functional consequences in adult blood. We did not find indications of differences between IVF and ICSI. STUDY FUNDING/COMPETING INTEREST(S): E.W.T. was supported by a VENI grant from the Netherlands Organization for Scientific Research (91617128) and JPI-H2020 Joint Programming Initiative a Healthy Diet for a Healthy Life (JPI HDHL) under proposal number 655 (PREcisE Project) through ZonMw (529051023). Financial support was provided from the European Union's Seventh Framework Program IDEAL (259679), the Swedish Research Council (K2011-69X-21871-01-6, 2011-3060, 2015-02434 and 2018-02640) and the Strategic Research Program in Epidemiology Young Scholar Awards, Karolinska Institute (to A.N.I.) and through the Swedish Initiative for Research on Microdata in the Social And Medical Sciences (SIMSAM) framework grant no 340-2013-5867, grants provided by the Stockholm County Council (ALF-projects), the Strategic Research Program in Epidemiology at Karolinska Institutet and the Swedish Heart-Lung Foundation and Danderyd University Hospital (Stockholm, Sweden). The funders had no role in study design, data collection, analysis, decision to publish or preparation of the manuscript. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

RNA-Seq in 296 phased trios provides a high-resolution map of genomic imprinting.

BACKGROUND: Identification of imprinted genes, demonstrating a consistent preference towards the paternal or maternal allelic expression, is important for the understanding of gene expression regulation during embryonic development and of the molecular basis of developmental disorders with a parent-of-origin effect. Combining allelic analysis of RNA-Seq data with phased genotypes in family trios provides a powerful method to detect parent-of-origin biases in gene expression. RESULTS: We report findings in 296 family trios from two large studies: 165 lymphoblastoid cell lines from the 1000 Genomes Project and 131 blood samples from the Genome of the Netherlands (GoNL) participants. Based on parental haplotypes, we identified > 2.8 million transcribed heterozygous SNVs phased for parental origin and developed a robust statistical framework for measuring allelic expression. We identified a total of 45 imprinted genes and one imprinted unannotated transcript, including multiple imprinted transcripts showing incomplete parental expression bias that was located adjacent to strongly imprinted genes. For example, PXDC1, a gene which lies adjacent to the paternally expressed gene FAM50B, shows a 2:1 paternal expression bias. Other imprinted genes had promoter regions that coincide with sites of parentally biased DNA methylation identified in the blood from uniparental disomy (UPD) samples, thus providing independent validation of our results. Using the stranded nature of the RNA-Seq data in lymphoblastoid cell lines, we identified multiple loci with overlapping sense/antisense transcripts, of which one is expressed paternally and the other maternally. Using a sliding window approach, we searched for imprinted expression across the entire genome, identifying a novel imprinted putative lncRNA in 13q21.2. Overall, we identified 7 transcripts showing parental bias in gene expression which were not reported in 4 other recent RNA-Seq studies of imprinting. CONCLUSIONS: Our methods and data provide a robust and high-resolution map of imprinted gene expression in the human genome.

Tea and coffee consumption in relation to DNA methylation in four European cohorts.

Lifestyle factors, such as food choices and exposure to chemicals, can alter DNA methylation and lead to changes in gene activity. Two such exposures with pharmacologically active components are coffee and tea consumption. Both coffee and tea have been suggested to play an important role in modulating disease-risk in humans by suppressing tumour progression, decreasing inflammation and influencing estrogen metabolism. These mechanisms may be mediated by changes in DNA methylation. To investigate if DNA methylation in blood is associated with coffee and tea consumption, we performed a genome-wide DNA methylation study for coffee and tea consumption in four European cohorts (N = 3,096). DNA methylation was measured from whole blood at 421,695 CpG sites distributed throughout the genome and analysed in men and women both separately and together in each cohort. Meta-analyses of the results and additional regional-level analyses were performed. After adjusting for multiple testing, the meta-analysis revealed that two individual CpG-sites, mapping to DNAJC16 and TTC17, were differentially methylated in relation to tea consumption in women. No individual sites were associated with men or with the sex-combined analysis for tea or coffee. The regional analysis revealed that 28 regions were differentially methylated in relation to tea consumption in women. These regions contained genes known to interact with estradiol metabolism and cancer. No significant regions were found in the sex-combined and male-only analysis for either tea or coffee consumption.

omicsPrint: detection of data linkage errors in multiple omics studies.

Summary: OmicsPrint is a versatile method for the detection of data linkage errors in multiple omics studies encompassing genetic, transcriptome and/or methylome data. OmicsPrint evaluates data linkage within and between omics data types using genotype calls from SNP arrays, DNA- or RNA-sequencing data and includes an algorithm to infer genotypes from Illumina DNA methylation array data. The method uses classification to verify assumed relationships and detect any data linkage errors, e.g. arising from sample mix-ups and mislabeling. Graphical and text output is provided to inspect and resolve putative data linkage errors. If sufficient genotype calls are available, first degree family relations also are revealed which can be used to check parent-offspring relations or zygosity in twin studies. Availability and implementation: omicsPrint is available from BioConductor; http://bioconductor.org/packages/omicsPrint. Supplementary information: Supplementary data are available at Bioinformatics online.

From promises to practical strategies in epigenetic epidemiology.

The epigenome has been heralded as a key 'missing piece' of the aetiological puzzle for complex phenotypes across the biomedical sciences. The standard research approaches developed for genetic epidemiology, however, are not necessarily appropriate for epigenetic studies of common disease. Here, we discuss the optimal execution of population-based studies of epigenetic variation, which will contribute to the emerging field of 'epigenetic epidemiology' and emphasize the importance of establishing a causal role in pathology for disease-associated epigenetic changes. We propose that improved understanding of the molecular mechanisms underlying human health and disease are best achieved through carrying out studies of epigenetics in populations as a part of an integrated functional genomics strategy.

TwinLIFE: The Twin Longitudinal Investigation of FEtal Discordance.

Lifelong health is thought to be partially set during intrauterine life by persistent epigenetic changes induced by the prenatal environment. To evaluate this hypothesis, we initiated a prospective longitudinal study in monochorionic (MC) twins: the TwinLIFE study. MC twins are monozygotic, thus in origin genetically identical, and share a single placenta. Although MC twins have many environmental factors in common, in one-third of the MC twin pairs, one fetus has significantly less access to nutrients and resources during pregnancy than its co-twin often resulting in a significant discordance in prenatal growth. Hence, MC twins constitute a unique natural experiment to study the influence of the prenatal environment on health. In TwinLIFE, we will chart intrapair differences in DNA methylation focusing on mesenchymal stromal cells isolated from cord as an advanced proxy of epigenetic dysregulation relevant for long-term health consequences. Next, we will follow up the MC twins for growth, cardiovascular and neurodevelopmental outcomes during childhood and evaluate the impact of an epigenetic signature at birth on future health. The current target is to include 100 MC twin pairs, but we aim to continue enrollment after procuring additional funding. TwinLIFE will not only address an unmet clinical need in the high-risk group of MC twins, but may also advance early-life strategies to prevent adverse growth, cardiovascular and neurodevelopmental outcomes in the general population.

IgG glycosylation and DNA methylation are interconnected with smoking.

BACKGROUND: Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic. METHODS: With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed. RESULTS: The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites. CONCLUSION: Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures. GENERAL SIGNIFICANCE: An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

DNA Methylation Landscapes of Human Fetal Development.

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.

The multifaceted interplay between lipids and epigenetics.

PURPOSE OF REVIEW: The interplay between lipids and epigenetic mechanisms has recently gained increased interest because of its relevance for common diseases and most notably atherosclerosis. This review discusses recent advances in unravelling this interplay with a particular focus on promising approaches and methods that will be able to establish causal relationships. RECENT FINDINGS: Complementary approaches uncovered close links between circulating lipids and epigenetic mechanisms at multiple levels. A characterization of lipid-associated genetic variants suggests that these variants exert their influence on lipid levels through epigenetic changes in the liver. Moreover, exposure of monocytes to lipids persistently alters their epigenetic makeup resulting in more proinflammatory cells. Hence, epigenetic changes can both impact on and be induced by lipids. SUMMARY: It is the combined application of technological advances to probe epigenetic modifications at a genome-wide scale and methodological advances aimed at causal inference (including Mendelian randomization and integrative genomics) that will elucidate the interplay between circulating lipids and epigenetics. Understanding its role in the development of atherosclerosis holds the promise of identifying a new category of therapeutic targets, since epigenetic changes are amenable to reversal.

Genome-wide association meta-analysis of human longevity identifies a novel locus conferring survival beyond 90 years of age.

The genetic contribution to the variation in human lifespan is ∼ 25%. Despite the large number of identified disease-susceptibility loci, it is not known which loci influence population mortality. We performed a genome-wide association meta-analysis of 7729 long-lived individuals of European descent (≥ 85 years) and 16 121 younger controls (<65 years) followed by replication in an additional set of 13 060 long-lived individuals and 61 156 controls. In addition, we performed a subset analysis in cases aged ≥ 90 years. We observed genome-wide significant association with longevity, as reflected by survival to ages beyond 90 years, at a novel locus, rs2149954, on chromosome 5q33.3 (OR = 1.10, P = 1.74 × 10(-8)). We also confirmed association of rs4420638 on chromosome 19q13.32 (OR = 0.72, P = 3.40 × 10(-36)), representing the TOMM40/APOE/APOC1 locus. In a prospective meta-analysis (n = 34 103), the minor allele of rs2149954 (T) on chromosome 5q33.3 associates with increased survival (HR = 0.95, P = 0.003). This allele has previously been reported to associate with low blood pressure in middle age. Interestingly, the minor allele (T) associates with decreased cardiovascular mortality risk, independent of blood pressure. We report on the first GWAS-identified longevity locus on chromosome 5q33.3 influencing survival in the general European population. The minor allele of this locus associates with low blood pressure in middle age, although the contribution of this allele to survival may be less dependent on blood pressure. Hence, the pleiotropic mechanisms by which this intragenic variation contributes to lifespan regulation have to be elucidated.

An alternative approach to multiple testing for methylation QTL mapping reduces the proportion of falsely identified CpGs.

INTRODUCTION: An increasing number of studies investigates the influence of local genetic variation on DNA methylation levels, so-called in cis methylation quantitative trait loci (meQTLs). A common multiple testing approach in genome-wide cis meQTL studies limits the false discovery rate (FDR) among all CpG-SNP pairs to 0.05 and reports on CpGs from the significant CpG-SNP pairs. However, a statistical test for each CpG is not performed, potentially increasing the proportion of CpGs falsely reported on. Here, we presented an alternative approach that properly control for multiple testing at the CpG level. RESULTS: We performed cis meQTL mapping for varying window sizes using publicly available single-nucleotide polymorphism (SNP) and 450 kb data, extracting the CpGs from the significant CpG-SNP pairs ([Formula: see text]). Using a new bait-and-switch simulation approach, we show that up to 50% of the CpGs found in the simulated data may be false-positive results. We present an alternative two-step multiple testing approach using the Simes and Benjamini-Hochberg procedures that does control the FDR among the CpGs, as confirmed by the bait-and-switch simulation. This approach indicates the use of window sizes in cis meQTL mapping studies that are significantly smaller than commonly adopted. DISCUSSION: Our approach to cis meQTL mapping properly controls the FDR at the CpG level, is computationally fast and can also be applied to cis eQTL studies. AVAILABILITY AND IMPLEMENTATION: An examplary R script for performing the Simes procedure is available as supplementary material. CONTACT: e.w.van_zwet@lumc.nl or b.t.heijmans@lumc.nl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

MethylAid: visual and interactive quality control of large Illumina 450k datasets.

UNLABELLED: The Illumina 450k array is a frequently used platform for large-scale genome-wide DNA methylation studies, i.e. epigenome-wide association studies. Currently, quality control of 450k data can be performed with Illumina's GenomeStudio and is part of a limited number 450k analysis pipelines. However, GenomeStudio cannot handle large-scale studies, and existing pipelines provide limited options for quality control and neither support interactive exploration by the user. To aid the detection of bad-quality samples in large-scale genome-wide DNA methylation studies as flexible and transparent as possible, we have developed MethylAid; a visual and interactive Web application using RStudio's shiny package. Bad-quality samples are detected using sample-dependent and sample-independent quality control probes present on the array and user-adjustable thresholds. In-depth exploration of bad-quality samples can be performed using several interactive diagnostic plots. Furthermore, plots can be annotated with user-provided metadata, for example, to identify outlying batches. Our new tool makes quality assessment of 450k array data interactive, flexible and efficient and is, therefore, expected to be useful for both data analysts and core facilities. AVAILABILITY AND IMPLEMENTATION: MethylAid is implemented as an R/Bioconductor package (www.bioconductor.org/packages/3.0/bioc/html/MethylAid.html). A demo application is available from shiny.bioexp.nl/MethylAid.