DNA-Methylation Signatures of Tobacco Smoking in a High Cardiovascular Risk Population: Modulation by the Mediterranean Diet.

Biomarkers based on DNA methylation are relevant in the field of environmental health for precision health. Although tobacco smoking is one of the factors with a strong and consistent impact on DNA methylation, there are very few studies analyzing its methylation signature in southern European populations and none examining its modulation by the Mediterranean diet at the epigenome-wide level. We examined blood methylation smoking signatures on the EPIC 850 K array in this population (n = 414 high cardiovascular risk subjects). Epigenome-wide methylation studies (EWASs) were performed analyzing differential methylation CpG sites by smoking status (never, former, and current smokers) and the modulation by adherence to a Mediterranean diet score was explored. Gene-set enrichment analysis was performed for biological and functional interpretation. The predictive value of the top differentially methylated CpGs was analyzed using receiver operative curves. We characterized the DNA methylation signature of smoking in this Mediterranean population by identifying 46 differentially methylated CpGs at the EWAS level in the whole population. The strongest association was observed at the cg21566642 (p = 2.2 × 10-32) in the 2q37.1 region. We also detected other CpGs that have been consistently reported in prior research and discovered some novel differentially methylated CpG sites in subgroup analyses. In addition, we found distinct methylation profiles based on the adherence to the Mediterranean diet. Particularly, we obtained a significant interaction between smoking and diet modulating the cg5575921 methylation in the AHRR gene. In conclusion, we have characterized biomarkers of the methylation signature of tobacco smoking in this population, and suggest that the Mediterranean diet can increase methylation of certain hypomethylated sites.

Single and Combined Associations of Plasma and Urine Essential Trace Elements (Zn, Cu, Se, and Mn) with Cardiovascular Risk Factors in a Mediterranean Population.

Trace elements are micronutrients that are required in very small quantities through diet but are crucial for the prevention of acute and chronic diseases. Despite the fact that initial studies demonstrated inverse associations between some of the most important essential trace elements (Zn, Cu, Se, and Mn) and cardiovascular disease, several recent studies have reported a direct association with cardiovascular risk factors due to the fact that these elements can act as both antioxidants and pro-oxidants, depending on several factors. This study aims to investigate the association between plasma and urine concentrations of trace elements and cardiovascular risk factors in a general population from the Mediterranean region, including 484 men and women aged 18−80 years and considering trace elements individually and as joint exposure. Zn, Cu, Se, and Mn were determined in plasma and urine using an inductively coupled plasma mass spectrometer (ICP-MS). Single and combined analysis of trace elements with plasma lipid, blood pressure, diabetes, and anthropometric variables was undertaken. Principal component analysis, quantile-based g-computation, and calculation of trace element risk scores (TERS) were used for the combined analyses. Models were adjusted for covariates. In single trace element models, we found statistically significant associations between plasma Se and increased total cholesterol and systolic blood pressure; plasma Cu and increased triglycerides and body mass index; and urine Zn and increased glucose. Moreover, in the joint exposure analysis using quantile g-computation and TERS, the combined plasma levels of Zn, Cu, Se (directly), and Mn (inversely) were strongly associated with hypercholesterolemia (OR: 2.03; 95%CI: 1.37−2.99; p < 0.001 per quartile increase in the g-computation approach). The analysis of urine mixtures revealed a significant relationship with both fasting glucose and diabetes (OR: 1.91; 95%CI: 1.01−3.04; p = 0.046). In conclusion, in this Mediterranean population, the combined effect of higher plasma trace element levels (primarily Se, Cu, and Zn) was directly associated with elevated plasma lipids, whereas the mixture effect in urine was primarily associated with plasma glucose. Both parameters are relevant cardiovascular risk factors, and increased trace element exposures should be considered with caution.

Association between Microbiome-Related Human Genetic Variants and Fasting Plasma Glucose in a High-Cardiovascular-Risk Mediterranean Population.

Background and Objectives: The gut microbiota has been increasingly recognized as a relevant factor associated with metabolic diseases. However, directly measuring the microbiota composition is a limiting factor for several studies. Therefore, using genetic variables as proxies for the microbiota composition is an important issue. Landmark microbiome-host genome-wide association studies (mbGWAS) have identified many SNPs associated with gut microbiota. Our aim was to analyze the association between relevant microbiome-related genetic variants (Mi-RSNPs) and fasting glucose and type 2 diabetes in a Mediterranean population, exploring the interaction with Mediterranean diet adherence. Materials and Methods: We performed a cross-sectional study in a high-cardiovascular-risk Mediterranean population (n = 1020), analyzing the association of Mi-RSNPs (from four published mbGWAS) with fasting glucose and type 2 diabetes. A single-variant approach was used for fitting fasting glucose and type 2 diabetes to a multivariable regression model. In addition, a Mendelian randomization analysis with multiple variants was performed as a sub-study. Results: We obtained several associations between Mi-RSNPs and fasting plasma glucose involving gut Gammaproteobacteria_HB, the order Rhizobiales, the genus Rumminococcus torques group, and the genus Tyzzerella as the top ranked. For type 2 diabetes, we also detected significant associations with Mi-RSNPs related to the order Rhizobiales, the family Desulfovibrionaceae, and the genus Romboutsia. In addition, some Mi-RSNPs and adherence to Mediterranean diet interactions were detected. Lastly, the formal Mendelian randomization analysis suggested combined effects. Conclusions: Although the use of Mi-RSNPs as proxies of the microbiome is still in its infancy, and although this is the first study analyzing such associations with fasting plasma glucose and type 2 diabetes in a Mediterranean population, some interesting associations, as well as modulations, with adherence to the Mediterranean diet were detected in these high-cardiovascular-risk subjects, eliciting new hypotheses.

Influence of DNA-Polymorphisms in Selected Circadian Clock Genes on Clock Gene Expression in Subjects from the General Population and Their Association with Sleep Duration.

Background and Objectives: Circadian rhythms have an important implication in numerous physiological and metabolic processes, including the sleep/wake cycle. Inter-individual differences in factors associated with circadian system may be due to gene differences in gene expression. Although several studies have analyzed the association between DNA polymorphisms and circadian variables, the influence on gene expression has been poorly analyzed. Our goal was to analyze the association of genetic variations in the clock genes and the gene expression level. Materials and Methods: We carried out a cross-sectional study of 102 adults (50.9% women). RNA and DNA were isolated from blood and single-nucleotide polymorphisms (SNPs), and the main circadian clock genes were determined. Gene expression of CLOCK, PER1, and VRK2 genes was measured by Reverse-transcription polymerase chain reaction (RT-PCR). The association between the DNA-SNPs and gene expression was analyzed at the gene level. In addition, a polygenic risk score (PRS), including all the significant SNPs related to gene expression, was created for each gene. Multivariable model analysis was performed. Results: Sex-specific differences were detected in PER1 expression, with these being higher in women (p = 0.034). No significant differences were detected in clock genes expression and lifestyle variables. We observed a significant association between the ARNTL-rs7924734, ARNTL-rs10832027, VRK2- rs2678902 SNPs, and CLOCK gene expression; the PER3-rs228642 and PER3-rs10127838 were related to PER1 expression, and the ARNTL-rs10832027, ARNTL-rs11022778, and MNTR1B-rs10830963 were associated with VRK2 gene expression (p < 0.05). The specific PRS created was significantly associated with each of the gene expressions analyzed (p < 0.001). Finally, sleep duration was associated with PER3-rs238666 (p = 0.008) and CLOCK-rs4580704 (p = 0.023). Conclusion: We detected significant associations between DNA-SNPs in the clock genes and their gene expression level in leukocytes and observed some differences in gene expression per sex. Moreover, we reported for the first time an association between clock gene polymorphisms and CLOCK, PER1, and VRK2 gene expression. These findings need further investigation.