Streptococcus Species Abundance in the Gut Is Linked to Subclinical Coronary Atherosclerosis in 8973 Participants From the SCAPIS Cohort.

BACKGROUND: Gut microbiota have been implicated in atherosclerotic disease, but their relation with subclinical coronary atherosclerosis is unclear. This study aimed to identify associations between the gut microbiome and computed tomography-based measures of coronary atherosclerosis and to explore relevant clinical correlates. METHODS: We conducted a cross-sectional study of 8973 participants (50 to 65 years of age) without overt atherosclerotic disease from the population-based SCAPIS (Swedish Cardiopulmonary Bioimage Study). Coronary atherosclerosis was measured using coronary artery calcium score and coronary computed tomography angiography. Gut microbiota species abundance and functional potential were assessed with shotgun metagenomics sequencing of fecal samples, and associations with coronary atherosclerosis were evaluated with multivariable regression models adjusted for cardiovascular risk factors. Associated species were evaluated for association with inflammatory markers, metabolites, and corresponding species in saliva. RESULTS: The mean age of the study sample was 57.4 years, and 53.7% were female. Coronary artery calcification was detected in 40.3%, and 5.4% had at least 1 stenosis with >50% occlusion. Sixty-four species were associated with coronary artery calcium score independent of cardiovascular risk factors, with the strongest associations observed for Streptococcus anginosus and Streptococcus oralis subsp oralis (P<1×10-5). Associations were largely similar across coronary computed tomography angiography-based measurements. Out of the 64 species, 19 species, including streptococci and other species commonly found in the oral cavity, were associated with high-sensitivity C-reactive protein plasma concentrations, and 16 with neutrophil counts. Gut microbial species that are commonly found in the oral cavity were negatively associated with plasma indole propionate and positively associated with plasma secondary bile acids and imidazole propionate. Five species, including 3 streptococci, correlated with the same species in saliva and were associated with worse dental health in the Malmö Offspring Dental Study. Microbial functional potential of dissimilatory nitrate reduction, anaerobic fatty acid ß-oxidation, and amino acid degradation were associated with coronary artery calcium score. CONCLUSIONS: This study provides evidence of an association of a gut microbiota composition characterized by increased abundance of Streptococcus spp and other species commonly found in the oral cavity with coronary atherosclerosis and systemic inflammation markers. Further longitudinal and experimental studies are warranted to explore the potential implications of a bacterial component in atherogenesis.

Gaseous air pollutants and DNA methylation in a methylome-wide association study of an ethnically and environmentally diverse population of U.S. adults.

Epigenetic mechanisms may underlie air pollution-health outcome associations. We estimated gaseous air pollutant-DNA methylation (DNAm) associations using twelve subpopulations within Women's Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) cohorts (n = 8397; mean age 61.3 years; 83% female; 46% African-American, 46% European-American, 8% Hispanic/Latino). We used geocoded participant address-specific mean ambient carbon monoxide (CO), nitrogen oxides (NO2; NOx), ozone (O3), and sulfur dioxide (SO2) concentrations estimated over the 2-, 7-, 28-, and 365-day periods before collection of blood samples used to generate Illumina 450 k array leukocyte DNAm measurements. We estimated methylome-wide, subpopulation- and race/ethnicity-stratified pollutant-DNAm associations in multi-level, linear mixed-effects models adjusted for sociodemographic, behavioral, meteorological, and technical covariates. We combined stratum-specific estimates in inverse variance-weighted meta-analyses and characterized significant associations (false discovery rate; FDR<0.05) at Cytosine-phosphate-Guanine (CpG) sites without among-strata heterogeneity (PCochran's Q > 0.05). We attempted replication in the Cooperative Health Research in Region of Augsburg (KORA) study and Normative Aging Study (NAS). We observed a -0.3 (95% CI: -0.4, -0.2) unit decrease in percent DNAm per interquartile range (IQR, 7.3 ppb) increase in 28-day mean NO2 concentration at cg01885635 (chromosome 3; regulatory region 290 bp upstream from ZNF621; FDR = 0.03). At intragenic sites cg21849932 (chromosome 20; LIME1; intron 3) and cg05353869 (chromosome 11; KLHL35; exon 2), we observed a -0.3 (95% CI: -0.4, -0.2) unit decrease (FDR = 0.04) and a 1.2 (95% CI: 0.7, 1.7) unit increase (FDR = 0.04), respectively, in percent DNAm per IQR (17.6 ppb) increase in 7-day mean ozone concentration. Results were not fully replicated in KORA and NAS. We identified three CpG sites potentially susceptible to gaseous air pollution-induced DNAm changes near genes relevant for cardiovascular and lung disease. Further harmonized investigations with a range of gaseous pollutants and averaging durations are needed to determine the effect of gaseous air pollutants on DNA methylation and ultimately gene expression.

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.

Epigenome-wide association study identifies TXNIP gene associated with type 2 diabetes mellitus and sustained hyperglycemia.

Type 2 diabetes mellitus (DM) is an established risk factor for a wide range of vascular diseases, including ischemic stroke (IS). Glycated hemoglobin A1c (HbA1c), a marker for average blood glucose levels over the previous 12 weeks, is used as a measure of glycemic control and also as a diagnostic criterion for diabetes (HbA1c levels ≥ 6.5%). Epigenetic mechanisms, such as DNA methylation, may be associated with aging processes and with modulation of the risk of various pathologies, such as DM. Specifically, DNA methylation could be one of the mechanisms mediating the relation between DM and environmental exposures. Our goal was to identify new CpG methylation sites associated with DM. We performed a genome-wide methylation study in whole-blood DNA from an IS patient cohorts. Illumina HumanMethylation450 BeadChip array was used to measure DNA methylation in CpG sites. All statistical analyses were adjusted for sex, age, hyperlipidemia, body mass index (BMI), smoking habit and cell count. Findings were replicated in two independent cohorts, an IS cohort and a population-based cohort, using the same array. In the discovery phase (N = 355), we identified a CpG site, cg19693031 (located in the TXNIP gene) that was associated with DM (P = 1.17 × 10(-12)); this CpG was replicated in two independent cohorts (N = 167 and N = 645). Methylation of TXNIP was inversely and intensely associated with HbA1c levels (P = 7.3 × 10(-16)), specifically related to diabetic patients with poor control of glucose levels. We identified an association between the TXNIP gene and DM through epigenetic mechanisms, related to sustained hyperglycemia levels (HbA1c ≥ 7%).

Causal modeling in a multi-omic setting: insights from GAW20.

BACKGROUND: Increasingly available multilayered omics data on large populations has opened exciting analytic opportunities and posed unique challenges to robust estimation of causal effects in the setting of complex disease phenotypes. The GAW20 Causal Modeling Working Group has applied complementary approaches (eg, Mendelian randomization, structural equations modeling, Bayesian networks) to discover novel causal effects of genomic and epigenomic variation on lipid phenotypes, as well as to validate prior findings from observational studies. RESULTS: Two Mendelian randomization studies have applied novel approaches to instrumental variable selection in methylation data, identifying bidirectional causal effects of CPT1A and triglycerides, as well as of RNMT and C6orf42, on high-density lipoprotein cholesterol response to fenofibrate. The CPT1A finding also emerged in a Bayesian network study. The Mendelian randomization studies have implemented both existing and novel steps to account for pleiotropic effects, which were independently detected in the GAW20 data via a structural equation modeling approach. Two studies estimated indirect effects of genomic variation (via DNA methylation and/or correlated phenotypes) on lipid outcomes of interest. Finally, a novel weighted R2 measure was proposed to complement other causal inference efforts by controlling for the influence of outlying observations. CONCLUSIONS: The GAW20 contributions illustrate the diversity of possible approaches to causal inference in the multi-omic context, highlighting the promises and assumptions of each method and the benefits of integrating both across methods and across omics layers for the most robust and comprehensive insights into disease processes.

Hypermethylation of MIR21 in CD4+ T cells from patients with relapsing-remitting multiple sclerosis associates with lower miRNA-21 levels and concomitant up-regulation of its target genes.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system caused by genetic and environmental factors. DNA methylation, an epigenetic mechanism that controls genome activity, may provide a link between genetic and environmental risk factors. OBJECTIVE: We sought to identify DNA methylation changes in CD4+ T cells in patients with relapsing-remitting (RR-MS) and secondary-progressive (SP-MS) disease and healthy controls (HC). METHODS: We performed DNA methylation analysis in CD4+ T cells from RR-MS, SP-MS, and HC and associated identified changes with the nearby risk allele, smoking, age, and gene expression. RESULTS: We observed significant methylation differences in the VMP1/MIR21 locus, with RR-MS displaying higher methylation compared to SP-MS and HC. VMP1/MIR21 methylation did not correlate with a known MS risk variant in VMP1 or smoking but displayed a significant negative correlation with age and the levels of mature miR-21 in CD4+ T cells. Accordingly, RR-MS displayed lower levels of miR-21 compared to SP-MS, which might reflect differences in age between the groups, and healthy individuals and a significant enrichment of up-regulated miR-21 target genes. CONCLUSION: Disease-related changes in epigenetic marking of MIR21 in RR-MS lead to differences in miR-21 expression with a consequence on miR-21 target genes.

DNA Methylation and High-Density Lipoprotein Functionality-Brief Report: The REGICOR Study (Registre Gironi del Cor).

OBJECTIVE: The function of high-density lipoproteins (HDLs) may better reflect their atheroprotective role, compared with HDL-cholesterol levels. The association between DNA methylation and HDL function has not yet been established. APPROACH AND RESULTS: We designed an epigenome-wide association study including 645 individuals from the REGICOR study (Registre Gironi del Cor). We determined DNA methylation from peripheral blood cells using the HumanMethylation450 array. We analyzed HDL functionality by determining HDL cholesterol efflux capacity and HDL inflammatory index. We discovered 3 methylation sites located in HOXA3, PEX5, and PER3 related to cholesterol efflux capacity and 1 located in GABRR1 related to HDL inflammatory index. Using a candidate gene approach, we also found 2 methylation sites located in CMIP related to cholesterol efflux capacity. CONCLUSIONS: We identified 6 potential loci associated with HDL functionality in HOXA3, PEX5, PER3, CMIP, and GABRR1. Additional studies are warranted to validate these findings in other populations.

Accelerometer-based physical activity is associated with the gut microbiota in 8416 individuals in SCAPIS.

BACKGROUND: Previous population-based studies investigating the relationship between physical activity and the gut microbiota have relied on self-reported activity, prone to reporting bias. Here, we investigated the associations of accelerometer-based sedentary (SED), moderate-intensity (MPA), and vigorous-intensity (VPA) physical activity with the gut microbiota using cross-sectional data from the Swedish CArdioPulmonary bioImage Study. METHODS: In 8416 participants aged 50-65, time in SED, MPA, and VPA were estimated with hip-worn accelerometer. Gut microbiota was profiled using shotgun metagenomics of faecal samples. We applied multivariable regression models, adjusting for sociodemographic, lifestyle, and technical covariates, and accounted for multiple testing. FINDINGS: Overall, associations between time in SED and microbiota species abundance were in opposite direction to those for MPA or VPA. For example, MPA was associated with lower, while SED with higher abundance of Escherichia coli. MPA and VPA were associated with higher abundance of the butyrate-producers Faecalibacterium prausnitzii and Roseburia spp. We observed discrepancies between specific VPA and MPA associations, such as a positive association between MPA and Prevotella copri, while no association was detected for VPA. Additionally, SED, MPA and VPA were associated with the functional potential of the microbiome. For instance, MPA was associated with higher capacity for acetate synthesis and SED with lower carbohydrate degradation capacity. INTERPRETATION: Our findings suggest that sedentary and physical activity are associated with a similar set of gut microbiota species but in opposite directions. Furthermore, the intensity of physical activity may have specific effects on certain gut microbiota species. FUNDING: European Research Council, Swedish Heart-Lung Foundation, Swedish Research Council, Knut and Alice Wallenberg Foundation.

Association of physical activity with high-density lipoprotein functionality in a population-based cohort: the REGICOR study.

INTRODUCTION AND OBJECTIVES: To determine the dose-response association between current and past leisure-time physical activity (LTPA), total and at different intensities, and high-density lipoprotein (HDL) functionality parameters. METHODS: Study participants (n=642) were randomly drawn from a large population-based survey. Mean age of the participants was 63.2 years and 51.1% were women. The analysis included data from a baseline and a follow-up visit (median follow-up, 4 years). LTPA was assessed using validated questionnaires at both visits. Two main HDL functions were assessed: cholesterol efflux capacity and HDL antioxidant capacity, at the follow-up visit. Linear regression and linear additive models were used to assess the linear and nonlinear association between LTPA and HDL functionality. RESULTS: Total LTPA at follow-up showed an inverse and linear relationship between 0 and 400 METs x min/d with HDL antioxidant capacity (regression coefficient [beta]: -0.022; 95%CI, -0.030, -0.013), with a plateau above this threshold. The results were similar for moderate (beta: -0.028; 95%CI, -0.049, -0.007) and vigorous (beta: -0.025; 95%CI, -0.043, -0.007), but not for light-intensity LTPA. LTPA at follow-up was not associated with cholesterol efflux capacity. Baseline LTPA was not associated with any of the HDL functionality parameters analyzed. CONCLUSIONS: Current moderate and vigorous LTPA showed a nonlinear association with higher HDL antioxidant capacity. Maximal benefit was observed with low-intermediate doses of total LTPA (up to 400 METs x min/d). Our results agree with current recommendations for moderate-vigorous LTPA practice and suggest an association between PA and HDL functionality in the general population.

OSA Is Associated With the Human Gut Microbiota Composition and Functional Potential in the Population-Based Swedish CardioPulmonary bioImage Study.

BACKGROUND: OSA is a common sleep-breathing disorder linked to increased risk of cardiovascular disease. Intermittent upper airway obstruction and hypoxia, hallmarks of OSA, have been shown in animal models to induce substantial changes to the gut microbiota composition, and subsequent transplantation of fecal matter to other animals induced changes in BP and glucose metabolism. RESEARCH QUESTION: Does OSA in adults associate with the composition and functional potential of the human gut microbiota? STUDY DESIGN AND METHODS: We used respiratory polygraphy data from up to 3,570 individuals 50 to 64 years of age from the population-based Swedish Cardiopulmonary bioimage Study combined with deep shotgun metagenomics of fecal samples to identify cross-sectional associations between three OSA parameters covering apneas and hypopneas, cumulative sleep time in hypoxia, and number of oxygen desaturation events with gut microbiota composition. Data collection about potential confounders was based on questionnaires, onsite anthropometric measurements, plasma metabolomics, and linkage with the Swedish Prescribed Drug Register. RESULTS: We found that all three OSA parameters were associated with lower diversity of species in the gut. Furthermore, in multivariable-adjusted analysis, the OSA-related hypoxia parameters were associated with the relative abundance of 128 gut bacterial species, including higher abundance of Blautia obeum and Collinsella aerofaciens. The latter species was also independently associated with increased systolic BP. Furthermore, the cumulative time in hypoxia during sleep was associated with the abundance of genes involved in nine gut microbiota metabolic pathways, including propionate production from lactate. Finally, we observed two heterogeneous sets of plasma metabolites with opposite association with species positively and negatively associated with hypoxia parameters, respectively. INTERPRETATION: OSA-related hypoxia, but not the number of apneas/hypopneas, is associated with specific gut microbiota species and functions. Our findings lay the foundation for future research on the gut microbiota-mediated health effects of OSA.

Development of gut microbiota during the first 2 years of life.

Although development of microbiota in childhood has been linked to chronic immune-related conditions, early childhood determinants of microbiota development have not been fully elucidated. We used 16S rRNA sequencing to analyse faecal and saliva samples from 83 children at four time-points during their first 2 years of life and from their mothers. Our findings confirm that gut microbiota in infants have low diversity and highlight that some properties are shared with the oral microbiota, although inter-individual differences are present. A considerable convergence in gut microbiota composition was noted across the first 2 years of life, towards a more diverse adult-like microbiota. Mode of delivery accounted for some of the inter-individual variation in early childhood, but with a pronounced attenuation over time. Our study extends previous research with further characterization of the major shift in gut microbiota composition during the first 2 years of life.

An online atlas of human plasma metabolite signatures of gut microbiome composition.

Human gut microbiota produce a variety of molecules, some of which enter the bloodstream and impact health. Conversely, dietary or pharmacological compounds may affect the microbiota before entering the circulation. Characterization of these interactions is an important step towards understanding the effects of the gut microbiota on health. In this cross-sectional study, we used deep metagenomic sequencing and ultra-high-performance liquid chromatography linked to mass spectrometry for a detailed characterization of the gut microbiota and plasma metabolome, respectively, of 8583 participants invited at age 50 to 64 from the population-based Swedish CArdioPulmonary bioImage Study. Here, we find that the gut microbiota explain up to 58% of the variance of individual plasma metabolites and we present 997 associations between alpha diversity and plasma metabolites and 546,819 associations between specific gut metagenomic species and plasma metabolites in an online atlas ( https://gutsyatlas.serve.scilifelab.se/ ). We exemplify the potential of this resource by presenting novel associations between dietary factors and oral medication with the gut microbiome, and microbial species strongly associated with the uremic toxin p-cresol sulfate. This resource can be used as the basis for targeted studies of perturbation of specific metabolites and for identification of candidate plasma biomarkers of gut microbiota composition.

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.

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.

Carbohydrate and fat intake associated with risk of metabolic diseases through epigenetics of CPT1A.

BACKGROUND: Epigenome-wide association studies identified the cg00574958 DNA methylation site at the carnitine palmitoyltransferase-1A (CPT1A) gene to be associated with reduced risk of metabolic diseases (hypertriglyceridemia, obesity, type 2 diabetes, hypertension, metabolic syndrome), but the mechanism underlying these associations is unknown. OBJECTIVES: We aimed to elucidate whether carbohydrate and fat intakes modulate cg00574958 methylation and the risk of metabolic diseases. METHODS: We examined associations between carbohydrate (CHO) and fat (FAT) intake, as percentages of total diet energy, and the CHO/FAT ratio with CPT1A-cg00574958, and the risk of metabolic diseases in 3 populations (Genetics of Lipid Lowering Drugs and Diet Network, n = 978; Framingham Heart Study, n = 2331; and REgistre GIroní del COR study, n = 645) while adjusting for confounding factors. To understand possible causal effects of dietary intake on the risk of metabolic diseases, we performed meta-analysis, CPT1A transcription analysis, and mediation analysis with CHO and FAT intakes as exposures and cg00574958 methylation as the mediator. RESULTS: We confirmed strong associations of cg00574958 methylation with metabolic phenotypes (BMI, triglyceride, glucose) and diseases in all 3 populations. Our results showed that CHO intake and CHO/FAT ratio were positively associated with cg00574958 methylation, whereas FAT intake was negatively correlated with cg00574958 methylation. Meta-analysis further confirmed this strong correlation, with ß = 58.4 ± 7.27, P = 8.98 x 10-16 for CHO intake; ß = -36.4 ± 5.95, P = 9.96 x 10-10 for FAT intake; and ß = 3.30 ± 0.49, P = 1.48 x 10-11 for the CHO/FAT ratio. Furthermore, CPT1A mRNA expression was negatively associated with CHO intake, and positively associated with FAT intake, and metabolic phenotypes. Mediation analysis supports the hypothesis that CHO intake induces CPT1A methylation, hence reducing the risk of metabolic diseases, whereas FAT intake inhibits CPT1A methylation, thereby increasing the risk of metabolic diseases. CONCLUSIONS: Our results suggest that the proportion of total energy supplied by CHO and FAT can have a causal effect on the risk of metabolic diseases via the epigenetic status of CPT1A.Study registration at https://www.clinicaltrials.gov/: the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)-NCT01023750; and the Framingham Heart Study (FHS)-NCT00005121.

Soluble low-density lipoprotein receptor-related protein 1 as a biomarker of coronary risk: Predictive capacity and association with clinical events.

BACKGROUND AND AIMS: We aimed to determine whether circulating sLRP1 levels are associated with future coronary events and improve the predictive capacity of the REGICOR (Registre Gironí del Cor) risk function. METHODS: We conducted a case-cohort study based on the follow-up of the REGICOR population-based cohort. Of the 5,404 participants aged between 35 and 74 years, without previous history of cardiovascular disease, 117 subjects with angina or fatal or non-fatal myocardial infarction were included, and 512 individuals were randomly selected as a subcohort (including 14 patients who presented coronary events). sLRP1 levels were measured in basal plasma samples by commercial ELISA. Hazard ratio (HR) was estimated with Cox models adjusted for potential confounding factors. Discrimination and reclassification were analyzed with the c-index and the net reclassification index (NRI), respectively. A Mendelian randomization approach was used to explore the causality of the association between sLRP1 and coronary artery disease (CAD). RESULTS: The group of participants who presented a CAD event showed higher levels of sLRP1 than the subcohort (2.45 [0.43; 8.31] vs. 2.07 [0.40; 6.65] µg/mL, p < 0.001). sLRP1 was significantly associated with CAD events even after adjustment for confounding factors (adjusted HR per standard deviation = 1.30, 95% CI: 1.01-1.67, p = 0.039). sLRP1 did not increase the predictive capacity or improve cardiovascular risk stratification of the REGICOR function. The LRP1 genetic variants associated with CAD risk were not related to sLRP1 concentration. CONCLUSIONS: Plasma sLRP1 is independently associated with the incidence of coronary events, but it does not improve the predictive capacity of the REGICOR risk function.

Disentangling associations between DNA methylation and blood lipids: a Mendelian randomization approach.

BACKGROUND: DNA methylation is an epigenetic mechanism that has been proposed as a possible link between genetic and environmental determinants of disease. Prior studies reported robust associations between the methylation of specific cytosine-phosphate-guanine (CpG) sites and plasma lipids, namely triglycerides (TGs) and high-density lipoprotein cholesterol (HDL-C). However, the causality of the observed association remains elusive, hampered by weak instrumental variables for methylation status. AIM: We present a novel application of the elastic net approach to implement a bidirectional Mendelian randomization approach to inferring causal relationships between candidate CpGs and plasma lipids in GAW20 data. METHODS: We used DNA methylation, TGs, and HDL-C measured during the visit 2. Based on prior findings, we selected 5 methylation markers (cg00574958, cg07504977, cg06690548, cg19693031, and cg03717755) related to TGs, 2 markers (cg09572125 and cg02650017) related to HDL-C, and 2 markers (cg06500161 and cg11024682) related to both traits. We implemented an elastic net approach to improve the selection of the genetic instrument for the methylation markers, followed by bidirectional Mendelian randomization 2-stage least-squares regression. RESULTS: We observed causal effects of blood fasting TGs on the methylation levels of cg00574958 (CPT1A) and cg06690548 (SLC7A11). For cg00574958, our findings were also consistent with the reverse direction of association, that is, from CPT1A methylation to TGs. CONCLUSIONS: Current evidence does not rule out either direction of association between the methylation of the cg00574958 CPT1A locus and plasma TGs, highlighting the complexity of lipid homeostasis. We also demonstrated a novel approach to improve instrument selection in DNA methylation studies.