Integrating multiple lines of evidence to assess the effects of maternal BMI on pregnancy and perinatal outcomes.

BACKGROUND: Higher maternal pre-pregnancy body mass index (BMI) is associated with adverse pregnancy and perinatal outcomes. However, whether these associations are causal remains unclear. METHODS: We explored the relation of maternal pre-/early-pregnancy BMI with 20 pregnancy and perinatal outcomes by integrating evidence from three different approaches (i.e. multivariable regression, Mendelian randomisation, and paternal negative control analyses), including data from over 400,000 women. RESULTS: All three analytical approaches supported associations of higher maternal BMI with lower odds of maternal anaemia, delivering a small-for-gestational-age baby and initiating breastfeeding, but higher odds of hypertensive disorders of pregnancy, gestational hypertension, preeclampsia, gestational diabetes, pre-labour membrane rupture, induction of labour, caesarean section, large-for-gestational age, high birthweight, low Apgar score at 1 min, and neonatal intensive care unit admission. For example, higher maternal BMI was associated with higher risk of gestational hypertension in multivariable regression (OR = 1.67; 95% CI = 1.63, 1.70 per standard unit in BMI) and Mendelian randomisation (OR = 1.59; 95% CI = 1.38, 1.83), which was not seen for paternal BMI (OR = 1.01; 95% CI = 0.98, 1.04). Findings did not support a relation between maternal BMI and perinatal depression. For other outcomes, evidence was inconclusive due to inconsistencies across the applied approaches or substantial imprecision in effect estimates from Mendelian randomisation. CONCLUSIONS: Our findings support a causal role for maternal pre-/early-pregnancy BMI on 14 out of 20 adverse pregnancy and perinatal outcomes. Pre-conception interventions to support women maintaining a healthy BMI may reduce the burden of obstetric and neonatal complications. FUNDING: Medical Research Council, British Heart Foundation, European Research Council, National Institutes of Health, National Institute for Health Research, Research Council of Norway, Wellcome Trust.

DNA Methylation and Age-Independent Cardiovascular Risk, an Epigenome-Wide Approach: The REGICOR Study (REgistre GIroní del COR).

OBJECTIVE: The objectives of this study were to decipher whether age-independent cardiovascular risk is associated with DNA methylation at 5'-cytosine-phosphate-guanine-3' (CpG) level and to determine whether these differential methylation signatures are associated with the incidence of cardiovascular events. APPROACH AND RESULTS: We designed a 2-stage, cross-sectional, epigenome-wide association study. Age-independent cardiovascular risk calculation was based on vascular age and on the residuals of the relationship between age and cardiovascular risk. Blood DNA methylomes from 2 independent populations were profiled using the Infinium HumanMethylation450 BeadChip. The discovery stage of these studies was performed in the REGICOR cohort (REgistre GIroní del COR; n=645). Next, we validated the initial findings in the Framingham Offspring Study (n=2542). Eight CpGs located in 4 genes (AHRR, CPT1A, PPIF, and SBNO2) and 3 intergenic regions showed differential methylation in association with age-independent cardiovascular risk (P≤1.17×10-7). These CpGs explained 12.01% to 15.16% of the variability of age-independent cardiovascular risk in REGICOR and 7.51% to 8.53% in Framingham Offspring Study. Four of them were only related to smoking, 3 were related to smoking and body mass index, and 1 to diabetes mellitus, triglycerides levels, and body mass index (P≤7.81×10-4). In addition, we developed methylation risk scores based on these CpGs and observed an association between these scores and cardiovascular disease incidence (hazard ratio=1.32; 95% confidence interval: 1.16-1.51). CONCLUSIONS: Age-independent cardiovascular risk was related to different DNA methylation profiles, with 8 CpGs showing differential methylation patterns. Most of these CpGs were associated with smoking, and 3 of them were also related to body mass index. Risk scores based on these differential methylation patterns were associated with cardiovascular events and could be useful predictive indices.

Association between long-term air pollution exposure and DNA methylation: The REGICOR study.

INTRODUCTION: Limited evidence suggests that epigenetic mechanisms may partially mediate the adverse effects of air pollution on health. Our aims were to identify new genomic loci showing differential DNA methylation associated with long-term exposure to air pollution and to replicate loci previously identified in other studies. METHODS: A two-stage epigenome-wide association study was designed: 630 individuals from the REGICOR study were included in the discovery and 454 participants of the EPIC-Italy study in the validation stage. DNA methylation was assessed using the Infinium HumanMethylation450 BeadChip. NOX, NO2, PM10, PM2.5, PMcoarse, traffic intensity and traffic load exposure were measured according to the ESCAPE protocol. A systematic review was undertaken to identify those cytosine-phosphate-guanine (CpGs) associated with air pollution in previous studies and we screened for them in the discovery study. RESULTS: In the discovery stage of the epigenome-wide association study, 81 unique CpGs were associated with air pollution (p-value <10-5) but none of them were validated in the replication sample. Furthermore, we identified 15 CpGs in the systematic review showing differential methylation with a p-value fulfilling the Bonferroni criteria and 1673 CpGs fulfilling the false discovery rate criteria, all of which were related to PM2.5 or NO2. None of them was replicated in the discovery study, in which the top hits were located in an intergenic region on chromosome 1 (cg10893043, p-value = 6.79·10-5) and in the LRRC45 and PXK genes (cg05088605, p-value = 2.15·10-04; cg16560256, p-value = 2.23·10-04). CONCLUSIONS: Neither new genomic loci associated with long-term air pollution were identified, nor previously identified loci were replicated. Continued efforts to test this potential association are warranted.

RNA Aptamers as Molecular Tools to Study the Functionality of the Hepatitis C Virus CRE Region.

BACKGROUND: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization of a set of aptamers targeting the cis-acting replication element (CRE) of the HCV genome, an essential partner for viral replication and also involved in the regulation of protein synthesis. METHODS: Forty-four aptamers were tested for their ability to interfere with viral RNA synthesis in a subgenomic replicon system. Some of the most efficient inhibitors were further evaluated for their potential to affect the recruitment of the HCV RNA-dependent RNA polymerase (NS5B) and the viral translation in cell culture. RESULTS: Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with functional RNA domains of the CRE, yielding a decrease in the HCV RNA levels higher than 90%. Concomitantly, one of them also induced a significant increase in viral translation (>50%). The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE. CONCLUSIONS: Present findings confirm the potential of the CRE as an anti-HCV target and support the use of aptamers as molecular tools for investigating the functionality of RNA domains in viral genomes.

Blood DNA methylation signature of diet quality and association with cardiometabolic traits.

AIMS: Diet quality might influence cardiometabolic health through epigenetic changes, but this has been little investigated in adults. Our aims were to identify cytosine-phosphate-guanine (CpG) dinucleotides associated with diet quality by conducting an epigenome-wide association study (EWAS) based on blood DNA methylation (DNAm) and to assess how diet-related CpGs associate with inherited susceptibility to cardiometabolic traits: body mass index (BMI), systolic blood pressure (SBP), triglycerides, type 2 diabetes (T2D), and coronary heart disease (CHD). METHODS AND RESULTS: Meta-EWAS including 5274 participants in four cohorts from Spain, the USA, and the UK. We derived three dietary scores (exposures) to measure adherence to a Mediterranean diet, to a healthy plant-based diet, and to the Dietary Approaches to Stop Hypertension. Blood DNAm (outcome) was assessed with the Infinium arrays Human Methylation 450K BeadChip and MethylationEPIC BeadChip. For each diet score, we performed linear EWAS adjusted for age, sex, blood cells, smoking and technical variables, and BMI in a second set of models. We also conducted Mendelian randomization analyses to assess the potential causal relationship between diet-related CpGs and cardiometabolic traits. We found 18 differentially methylated CpGs associated with dietary scores (P < 1.08 × 10-7; Bonferroni correction), of which 12 were previously associated with cardiometabolic traits. Enrichment analysis revealed overrepresentation of diet-associated genes in pathways involved in inflammation and cardiovascular disease. Mendelian randomization analyses suggested that genetically determined methylation levels corresponding to lower diet quality at cg02079413 (SNORA54), cg02107842 (MAST4), and cg23761815 (SLC29A3) were causally associated with higher BMI and at cg05399785 (WDR8) with greater SBP, and methylation levels associated with higher diet quality at cg00711496 (PRMT1) with lower BMI, T2D risk, and CHD risk and at cg0557921 (AHRR) with lower CHD risk. CONCLUSION: Diet quality in adults was related to differential methylation in blood at 18 CpGs, some of which related to cardiometabolic health.

We conducted a study to investigate the connection between diet quality, epigenetic changes, and cardiovascular health in adults. The study included 5274 participants from Spain, the USA, and the UK, combining data from four different cohorts. We assessed adherence to different healthy diets: Mediterranean style diet, plant-based diet, and Dietary Approaches to Stop Hypertension diet. We used advanced technology to analyse blood DNA methylation, which refers to chemical modifications in the DNA that can affect gene activity.We discovered 18 CpGs that showed differential methylation patterns related to the dietary scores. Importantly, 12 of these CpGs had previously been associated with cardiovascular disease or risk factors, suggesting a potential link between diet, epigenetic changes, and heart health. Some of the diet-related CpGs mapped to genes involved in pathways associated with cardiovascular disease. Moreover, using a method called Mendelian randomization, we found that several CpGs may have a causal association with body mass index, systolic blood pressure, and risk of type 2 diabetes and coronary heart disease.

Case report of plastic nurdles pollution in Galicia (NW Atlantic) following the Toconao's spill in December 2023: The VIEIRA Collaborative.

Plastic nurdles pose a significant environmental threat due to recurrent accidental spills into marine ecosystems. This report examines the nurdle pollution over the 1498 km of the Galician coastline (Spain) following the spill of 25 t of nurdles into the Northwest Atlantic after the loss of six containers from the Toconao vessel in December 2023. This accident highlights the urgent need for proactive, effective measures in maritime transport to prevent and mitigate such environmental catastrophes. The complexity of nurdle dispersion challenges the evaluation of their fate at sea, and the potential long-term consequences on the marine ecosystem and food web remain uncertain and yet to be investigated. This report also presents the VIEIRA collaborative and underscores the critical role of citizen-led initiatives in responding to such environmental disasters, and advocates for efficient policy reforms, involving cross-border collaboration. Furthermore, we call for greater international cooperation to underpin effective regulatory frameworks to address the growing hazard of plastic nurdle pollution worldwide.

Exploring the impact of selection bias in observational studies of COVID-19: a simulation study.

BACKGROUND: Non-random selection of analytic subsamples could introduce selection bias in observational studies. We explored the potential presence and impact of selection in studies of SARS-CoV-2 infection and COVID-19 prognosis. METHODS: We tested the association of a broad range of characteristics with selection into COVID-19 analytic subsamples in the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK Biobank (UKB). We then conducted empirical analyses and simulations to explore the potential presence, direction and magnitude of bias due to this selection (relative to our defined UK-based adult target populations) when estimating the association of body mass index (BMI) with SARS-CoV-2 infection and death-with-COVID-19. RESULTS: In both cohorts, a broad range of characteristics was related to selection, sometimes in opposite directions (e.g. more-educated people were more likely to have data on SARS-CoV-2 infection in ALSPAC, but less likely in UKB). Higher BMI was associated with higher odds of SARS-CoV-2 infection and death-with-COVID-19. We found non-negligible bias in many simulated scenarios. CONCLUSIONS: Analyses using COVID-19 self-reported or national registry data may be biased due to selection. The magnitude and direction of this bias depend on the outcome definition, the true effect of the risk factor and the assumed selection mechanism; these are likely to differ between studies with different target populations. Bias due to sample selection is a key concern in COVID-19 research based on national registry data, especially as countries end free mass testing. The framework we have used can be applied by other researchers assessing the extent to which their results may be biased for their research question of interest.

Effects of the maternal and fetal proteome on birth weight: a Mendelian randomization analysis.

Fetal growth is an indicator of fetal survival, regulated by maternal and fetal factors, but little is known about the underlying molecular mechanisms. We used Mendelian randomization to explore the effects of maternal and fetal genetically-instrumented plasma proteins on birth weight using genome-wide association summary data (n=406,063 with maternal and/or fetal genotype), with independent replication (n=74,932 mothers and n=62,108 offspring), and colocalisation. Higher genetically-predicted maternal levels of PCSK1 increased birthweight (mean-difference: 9g (95% CI: 5g, 13g) per 1 standard deviation protein level). Higher maternal levels of LGALS4 decreased birthweight (-54g (-29g, -80g)), as did VCAM1, RAD51D and GP1BA. In the offspring, higher genetically-predicted fetal levels of LGALS4 (46g (23g, 70g)) increased birthweight, alongside FCGR2B. Higher offspring levels of PCSK1 decreased birth weight (-9g (-16g, 4g), alongside LEPR. Results support maternal and fetal protein effects on birth weight, implicating roles for glucose metabolism, energy homeostasis, endothelial function and adipocyte differentiation.

DNA Methylation and Ischemic Stroke Risk: An Epigenome-Wide Association Study.

BACKGROUND: Ischemic stroke (IS) risk heritability is partly explained by genetics. Other heritable factors, such as epigenetics, could explain an unknown proportion of the IS risk. The objective of this study is to evaluate DNA methylation association with IS using epigenome-wide association studies (EWAS). METHODS: We performed a two-stage EWAS comprising 1,156 subjects. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) were assessed using the Infinium 450K and EPIC BeadChip in the discovery cohort (252 IS and 43 controls). Significant DMPs were replicated in an independent cohort (618 IS and 243 controls). Stroke subtype associations were also evaluated. Differentially methylated cell-type (DMCT) was analyzed in the replicated CpG sites using EpiDISH. We additionally performed pathway enrichment analysis and causality analysis with Mendelian randomization for the replicated CpG sites. RESULTS: A total of 957 CpG sites were epigenome-wide-significant (p ≤ 10-7) in the discovery cohort, being CpG sites in the top signals (logFC = 0.058, p = 2.35 × 10-22; logFC = 0.035, p = 3.22 × 10-22, respectively). ZFHX3 and MAP3K1 were among the most significant DMRs. In addition, 697 CpG sites were replicated considering Bonferroni-corrected p-values (p < 5.22 × 10-5). All the replicated DMPs were associated with risk of cardioembolic, atherothrombotic, and undetermined stroke. The DMCT analysis demonstrated that the significant associations were driven by natural killer cells. The pathway enrichment analysis showed overrepresentation of genes belonging to certain pathways including oxidative stress. ZFHX3 and MAP3K1 methylation was causally associated with specific stroke-subtype risk. CONCLUSION: Specific DNA methylation pattern is causally associated with IS risk. These results could be useful for specifically predicting stroke occurrence and could potentially be evaluated as therapeutic targets.

DNA methylation and gene expression integration in cardiovascular disease.

BACKGROUND: The integration of different layers of omics information is an opportunity to tackle the complexity of cardiovascular diseases (CVD) and to identify new predictive biomarkers and potential therapeutic targets. Our aim was to integrate DNA methylation and gene expression data in an effort to identify biomarkers related to cardiovascular disease risk in a community-based population. We accessed data from the Framingham Offspring Study, a cohort study with data on DNA methylation (Infinium HumanMethylation450 BeadChip; Illumina) and gene expression (Human Exon 1.0 ST Array; Affymetrix). Using the MOFA2 R package, we integrated these data to identify biomarkers related to the risk of presenting a cardiovascular event. RESULTS: Four independent latent factors (9, 19, 21-only in women-and 27), driven by DNA methylation, were associated with cardiovascular disease independently of classical risk factors and cell-type counts. In a sensitivity analysis, we also identified factor 21 as associated with CVD in women. Factors 9, 21 and 27 were also associated with coronary heart disease risk. Moreover, in a replication effort in an independent study three of the genes included in factor 27 were also present in a factor identified to be associated with myocardial infarction (CDC42BPB, MAN2A2 and RPTOR). Factor 9 was related to age and cell-type proportions; factor 19 was related to age and B cells count; factor 21 pointed to human immunodeficiency virus infection-related pathways and inflammation; and factor 27 was related to lifestyle factors such as alcohol consumption, smoking and body mass index. Inclusion of factor 21 (only in women) improved the discriminative and reclassification capacity of the Framingham classical risk function and factor 27 improved its discrimination. CONCLUSIONS: Unsupervised multi-omics data integration methods have the potential to provide insights into the pathogenesis of cardiovascular diseases. We identified four independent factors (one only in women) pointing to inflammation, endothelium homeostasis, visceral fat, cardiac remodeling and lifestyles as key players in the determination of cardiovascular risk. Moreover, two of these factors improved the predictive capacity of a classical risk function.

Bias from questionnaire invitation and response in COVID-19 research: an example using ALSPAC.

Background: Longitudinal studies are crucial for identifying potential risk factors for infection with, and consequences of, COVID-19, but relationships can be biased if they are associated with invitation and response to data collection. We describe factors relating to questionnaire invitation and response in COVID-19 questionnaire data collection in a multigenerational birth cohort (the Avon Longitudinal Study of Parents and Children, ALSPAC). Methods: We analysed online questionnaires completed between the beginning of the pandemic and easing of the first UK lockdown by participants with valid email addresses who had not actively disengaged from the study. We assessed associations of pre-pandemic sociodemographic, behavioural, anthropometric and health-related factors with: i) being sent a questionnaire; ii) returning a questionnaire; and iii) item response (for specific questions). Analyses were conducted in three cohorts: the index children born in the early 1990s (now young adults; 41 variables assessed), their mothers (35 variables) and the mothers' partners (27 variables). Results: Of 14,849 young adults, 41% were sent a questionnaire, of whom 57% returned one. Item response was >95%. In this cohort, 78% of factors were associated with being sent a questionnaire, 56% with returning one, and, as an example of item response, 20% with keyworker status response. For instance, children from mothers educated to degree-level had greater odds of being sent a questionnaire (OR=5.59; 95% CI=4.87-6.41), returning one (OR=1.60; 95% CI=1.31-1.95), and responding to items (e.g., keyworker status OR=1.65; 95% CI=0.88-3.04), relative to children from mothers with fewer qualifications. Invitation and response rates and associations were similar in all cohorts. Conclusions: These results highlight the importance of considering potential biases due to non-response when using longitudinal studies in COVID-19 research and interpreting results. We recommend researchers report response rates and factors associated with invitation and response in all COVID-19 observational research studies, which can inform sensitivity analyses.

Analysis of the dose-response relationship of leisure-time physical activity to cardiovascular disease and all-cause mortality: the REGICOR study.

INTRODUCTION AND OBJECTIVES: Regular leisure-time physical activity (LTPA) has been consistently recognized as a protective factor for cardiovascular diseases (CVD) and all-cause mortality. However, the pattern of this relationship is still not clear. The aim of this study was to assess the relationship of LTPA with incident CVD and mortality in a Spanish population. METHODS: A prospective population-based cohort of 11 158 randomly selected inhabitants from the general population. LTPA was assessed by a validated questionnaire. Mortality and CVD outcomes were registered during the follow-up (median: 7.24 years). The association between LTPA and outcomes of interest (all-cause mortality and cardiovascular disease) was explored using a generalized additive model with penalized smoothing splines and multivariate Cox proportional hazard models. RESULTS: We observed a significant nonlinear association between LTPA and all-cause and CVD mortality, and fatal and nonfatal CVD. Moderate-vigorous intensity LTPA, but not light-intensity LTPA, were associated with beneficial effects. The smoothing splines identified a cutoff at 400 MET-min/d. Below this threshold, each increase of 100 MET-min/d in moderate-vigorous LTPA contributed with a 16% risk reduction in all-cause mortality (HR, 0.84; 95%CI, 0.77-0.91), a 27% risk reduction in CVD mortality (HR, 0.73; 95%CI, 0.61-0.87), and a 12% risk reduction in incident CVD (HR, 0.88; 95%CI, 0.79-0.99). No further benefits were observed beyond 400 MET-min/d. CONCLUSIONS: Our results support a nonlinear inverse relationship between moderate-vigorous LTPA and CVD and mortality. Benefits of PA are already observed with low levels of activity, with a maximum benefit around 3 to 5 times the current recommendations.

DNA methylation biomarkers of myocardial infarction and cardiovascular disease.

BACKGROUND: The epigenetic landscape underlying cardiovascular disease (CVD) is not completely understood and the clinical value of the identified biomarkers is still limited. We aimed to identify differentially methylated loci associated with acute myocardial infarction (AMI) and assess their validity as predictive and causal biomarkers. RESULTS: We designed a case-control, two-stage, epigenome-wide association study on AMI (ndiscovery = 391, nvalidation = 204). DNA methylation was assessed using the Infinium MethylationEPIC BeadChip. We performed a fixed-effects meta-analysis of the two samples. 34 CpGs were associated with AMI. Only 12 of them were available in two independent cohort studies (n ~ 1800 and n ~ 2500) with incident coronary and cardiovascular disease (CHD and CVD, respectively). The Infinium HumanMethylation450 BeadChip was used in those two studies. Four of the 12 CpGs were validated in association with incident CHD: AHRR-mapping cg05575921, PTCD2-mapping cg25769469, intergenic cg21566642 and MPO-mapping cg04988978. We then assessed whether methylation risk scores based on those CpGs improved the predictive capacity of the Framingham risk function, but they did not. Finally, we aimed to study the causality of those associations using a Mendelian randomization approach but only one of the CpGs had a genetic influence and therefore the results were not conclusive. CONCLUSIONS: We have identified 34 CpGs related to AMI. These loci highlight the relevance of smoking, lipid metabolism, and inflammation in the biological mechanisms related to AMI. Four were additionally associated with incident CHD and CVD but did not provide additional predictive information.

Epigenomic Assessment of Cardiovascular Disease Risk and Interactions With Traditional Risk Metrics.

Background Epigenome-wide association studies for cardiometabolic risk factors have discovered multiple loci associated with incident cardiovascular disease (CVD). However, few studies have sought to directly optimize a predictor of CVD risk. Furthermore, it is challenging to train multivariate models across multiple studies in the presence of study- or batch effects. Methods and Results Here, we analyzed existing DNA methylation data collected using the Illumina HumanMethylation450 microarray to create a predictor of CVD risk across 3 cohorts: Women's Health Initiative, Framingham Heart Study Offspring Cohort, and Lothian Birth Cohorts. We trained Cox proportional hazards-based elastic net regressions for incident CVD separately in each cohort and used a recently introduced cross-study learning approach to integrate these individual scores into an ensemble predictor. The methylation-based risk score was associated with CVD time-to-event in a held-out fraction of the Framingham data set (hazard ratio per SD=1.28, 95% CI, 1.10-1.50) and predicted myocardial infarction status in the independent REGICOR (Girona Heart Registry) data set (odds ratio per SD=2.14, 95% CI, 1.58-2.89). These associations remained after adjustment for traditional cardiovascular risk factors and were similar to those from elastic net models trained on a directly merged data set. Additionally, we investigated interactions between the methylation-based risk score and both genetic and biochemical CVD risk, showing preliminary evidence of an enhanced performance in those with less traditional risk factor elevation. Conclusions This investigation provides proof-of-concept for a genome-wide, CVD-specific epigenomic risk score and suggests that DNA methylation data may enable the discovery of high-risk individuals who would be missed by alternative risk metrics.

High-density lipoprotein characteristics and coronary artery disease: a Mendelian randomization study.

BACKGROUND: To assess whether genetically determined quantitative and qualitative HDL characteristics were independently associated with coronary artery disease (CAD). METHODS: We designed a two-sample multivariate Mendelian randomization study with available genome-wide association summary data. We identified genetic variants associated with HDL cholesterol and apolipoprotein A-I levels, HDL size, particle levels, and lipid content to define our genetic instrumental variables in one sample (Kettunen et al. study, n = 24,925) and analyzed their association with CAD risk in a different study (CARDIoGRAMplusC4D, n = 184,305). We validated these results by defining our genetic variables in another database (METSIM, n = 8372) and studied their relationship with CAD in the CARDIoGRAMplusC4D dataset. To estimate the effect size of the associations of interest adjusted for other lipoprotein traits and minimize potential pleiotropy, we used the Multi-trait-based Conditional & Joint analysis. RESULTS: Genetically determined HDL cholesterol and apolipoprotein A-I levels were not associated with CAD. HDL mean diameter (ß = 0.27 [95%CI = 0.19; 0.35]), cholesterol levels in very large HDLs (ß = 0.29 [95%CI = 0.17; 0.40]), and triglyceride content in very large HDLs (ß = 0.14 [95%CI = 0.040; 0.25]) were directly associated with CAD risk, whereas the cholesterol content in medium-sized HDLs (ß = -0.076 [95%CI = -0.10; -0.052]) was inversely related to this risk. These results were validated in the METSIM-CARDIoGRAMplusC4D data. CONCLUSIONS: Some qualitative HDL characteristics (related to size, particle distribution, and cholesterol and triglyceride content) are related to CAD risk while HDL cholesterol levels are not.

Physical Activity and Genome-wide DNA Methylation: The REgistre GIroní del COR Study.

INTRODUCTION: DNA methylation may be one of the biological mechanisms underlying the health benefits of physical activity (PA). Our objective was to determine the association between PA and genome-wide DNA methylation at CpG level. METHODS: We designed a two-stage epigenome wide association study. In the discovery stage, we used 619 individuals from the REgistre GIroní del COR cohort. Next, we validated the CpG suggestively associated with PA (P < 10) in two independent populations (n = 1735 and 190, respectively). Physical activity was assessed with validated questionnaires and classified as light PA (LPA), moderate PA, vigorous PA, moderate-vigorous PA (MVPA) and total PA. We examined linear and nonlinear associations and meta-analyzed the results in the three populations. The linear associations were meta-analyzed with a fixed-effects model and the P values of the nonlinear associations with the Stouffer and Fisher methods. We established a P value threshold that fulfilled Bonferroni criteria over the number of CpG analyzed (0.05/421,940 = 1.185 × 10). RESULTS: In the meta-analyses, two CpG sites had a statistically significant nonlinear association with MVPA. cg24155427 (P = 1.19 × 10), located in an intergenic region in chromosome 1, has been previously associated with smoking, lupus, and aging. cg09565397 (P = 1.59 × 10), located within DGAT1 in chromosome 8, which encodes an enzyme involved in triacylglycerol synthesis. CONCLUSIONS: This population-based study identified two new, differentially methylated CpG sites with a nonlinear dose-response relationship to MVPA. These associations must be additionally validated and may be considered for further research on the biological mechanisms underlying health benefits of PA.

DNA methylation of MMPs and TIMPs in atherothrombosis process in carotid plaques and blood tissues.

BACKGROUND AND PURPOSE: Polymorphisms and serum levels of Matrix Metalloproteinases (MMP) and Tissue Inhibitor of Metalloproteinases (TIMP) have been studied with regard to atheromatous plaques and ischemic stroke, while no studies of DNA methylation (DNAm) patterns of MMP or TIMP have been performed to that end. Here, we evaluate DNAm levels of the MMP and TIMP gene families in human carotid plaques and blood samples of atherothrombotic stroke patients. METHODS: We profiled the DNAm status of stable and ulcerated atherosclerotic plaques obtained as pair sets from three patients who underwent carotid endarterectomy surgery. We selected 415 CpG sites, mapping into MMPs and TIMPs genes for further study. Secondly, the statistically associated CpG sites were analyzed in blood samples from two separate atherothrombotic stroke cohorts (total sample size = 307), ischemic stroke-cohort 1 (ISC-1): 37 atherothrombotic patients and 6 controls, ischemic stroke-cohort 2 (ISC-2): 80 atherothrombotic patients and 184 controls. DNAm levels from plaque tissue and blood samples were evaluated using a high-density microarray Infinium, HumanMethylation450 BeadChip and Infinium MethylationEPIC BeadChip. RESULTS: Three CpG sites were statistically significantly associated with unstable plaque portions; cg02969624, q-value = 0.035 (TIMP2), and cg04316754, q-value = 0.037 (MMP24) were hypermethylated, while cg24211657 q-value = 0.035 (TIMP2) was hypomethylated. Association of cg04316754 (MMP24) methylation levels with atherothrombotic risk was also observed in blood tissue: ISC-1 p-values = 0.03, ISC-2 p-value = 1.9 × 10-04. CONCLUSIONS: The results suggest different DNAm status of MMP24 between stable and unstable atherothrombotic carotid plaques, and between atherothrombotic stroke and controls in blood samples.

Identification of 20 novel loci associated with ischaemic stroke. Epigenome-wide association study.

DNA methylation is dynamic, varies throughout the life course, and its levels are influenced by lifestyle and environmental factors, as well as by genetic variation. The leading genetic variants at stroke risk loci identified to date explain roughly 1-2% of stroke heritability. Most of these single nucleotide polymorphisms are situated within a regulatory sequence marked by DNase I hypersensitivity sites, which would indicate involvement of an epigenetic mechanism. To detect epigenetic variants associated with stroke occurrence and stroke subtypes. A two-stage case-control epigenome-wide association study was designed. The discovery sample with 401 samples included 218 ischaemic stroke (IS) patients, assessed at Hospital del Mar (Barcelona, Spain) and 183 controls from the REGICOR cohort. In two independent samples (N = 226 and N = 166), we replicated 22 CpG sites differentially methylated in IS in 21 loci, including 2 CpGs in locus ZFHX3, which includes known genetic variants associated with stroke. The pathways associated with these loci are inflammation and angiogenesis. The meta-analysis identified 384 differentially methylated CpGs, including loci of known stroke and vascular risk genetic variants, enriched by loci involved in lipid metabolism, adipogenesis, circadian clock, and glycolysis pathways. We identified a set of 22 CpGs in 21 loci associated with IS. Our analysis suggests that DNA methylation changes may contribute to orchestrating gene expression that contributes to IS.

Functional Information Stored in the Conserved Structural RNA Domains of Flavivirus Genomes.

The genus Flavivirus comprises a large number of small, positive-sense single-stranded, RNA viruses able to replicate in the cytoplasm of certain arthropod and/or vertebrate host cells. The genus, which has some 70 member species, includes a number of emerging and re-emerging pathogens responsible for outbreaks of human disease around the world, such as the West Nile, dengue, Zika, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Like other RNA viruses, flaviviruses have a compact RNA genome that efficiently stores all the information required for the completion of the infectious cycle. The efficiency of this storage system is attributable to supracoding elements, i.e., discrete, structural units with essential functions. This information storage system overlaps and complements the protein coding sequence and is highly conserved across the genus. It therefore offers interesting potential targets for novel therapeutic strategies. This review summarizes our knowledge of the features of flavivirus genome functional RNA domains. It also provides a brief overview of the main achievements reported in the design of antiviral nucleic acid-based drugs targeting functional genomic RNA elements.

DNA methylation and obesity traits: An epigenome-wide association study. The REGICOR study.

Obesity is associated with increased risk of several diseases and has become epidemic. Obesity is highly heritable but the genetic variants identified by genome-wide association studies explain only limited variability. Epigenetics could contribute to explain the missing variability. The study aim was to discover differential methylation patterns related to obesity. We designed an epigenome-wide association study with a discovery phase in a subsample of 641 REGICOR study participants, validated by analysis of 2,515 participants in the Framingham Offspring Study. Blood DNA methylation was assessed using Illumina HumanMethylation450 BeadChip. Next, we meta-analyzed the data using the fixed effects method and performed a functional and pathway analysis using the Ingenuity Pathway Analysis software. We were able to validate 94 CpGs associated with body mass index (BMI) and 49 CpGs associated with waist circumference, located in 95 loci. In addition, we newly discovered 70 CpGs associated with BMI and 33 CpGs related to waist circumference. These CpGs explained 25.94% and 29.22% of the variability of BMI and waist circumference, respectively, in the REGICOR sample. We also evaluated 65 of the 95 validated loci in the GIANT genome-wide association data; 10 of them had Tag SNPs associated with BMI. The top-ranked diseases and functions identified in the functional and pathway analysis were neurologic, psychological, endocrine, and metabolic.