Genetic analysis of over half a million people characterises C-reactive protein loci.

Chronic low-grade inflammation is linked to a multitude of chronic diseases. We report the largest genome-wide association study (GWAS) on C-reactive protein (CRP), a marker of systemic inflammation, in UK Biobank participants (N = 427,367, European descent) and the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium (total N = 575,531 European descent). We identify 266 independent loci, of which 211 are not previously reported. Gene-set analysis highlighted 42 gene sets associated with CRP levels (p ≤ 3.2 ×10-6) and tissue expression analysis indicated a strong association of CRP related genes with liver and whole blood gene expression. Phenome-wide association study identified 27 clinical outcomes associated with genetically determined CRP and subsequent Mendelian randomisation analyses supported a causal association with schizophrenia, chronic airway obstruction and prostate cancer. Our findings identified genetic loci and functional properties of chronic low-grade inflammation and provided evidence for causal associations with a range of diseases.

Meta-analysis of epigenome-wide association studies of carotid intima-media thickness.

Common carotid intima-media thickness (cIMT) is an index of subclinical atherosclerosis that is associated with ischemic stroke and coronary artery disease (CAD). We undertook a cross-sectional epigenome-wide association study (EWAS) of measures of cIMT in 6400 individuals. Mendelian randomization analysis was applied to investigate the potential causal role of DNA methylation in the link between atherosclerotic cardiovascular risk factors and cIMT or clinical cardiovascular disease. The CpG site cg05575921 was associated with cIMT (beta = -0.0264, p value = 3.5 × 10-8) in the discovery panel and was replicated in replication panel (beta = -0.07, p value = 0.005). This CpG is located at chr5:81649347 in the intron 3 of the aryl hydrocarbon receptor repressor gene (AHRR). Our results indicate that DNA methylation at cg05575921 might be in the pathway between smoking, cIMT and stroke. Moreover, in a region-based analysis, 34 differentially methylated regions (DMRs) were identified of which a DMR upstream of ALOX12 showed the strongest association with cIMT (p value = 1.4 × 10-13). In conclusion, our study suggests that DNA methylation may play a role in the link between cardiovascular risk factors, cIMT and clinical cardiovascular disease.

Blood Leukocyte DNA Methylation Predicts Risk of Future Myocardial Infarction and Coronary Heart Disease.

BACKGROUND: DNA methylation is implicated in coronary heart disease (CHD), but current evidence is based on small, cross-sectional studies. We examined blood DNA methylation in relation to incident CHD across multiple prospective cohorts. METHODS: Nine population-based cohorts from the United States and Europe profiled epigenome-wide blood leukocyte DNA methylation using the Illumina Infinium 450k microarray, and prospectively ascertained CHD events including coronary insufficiency/unstable angina, recognized myocardial infarction, coronary revascularization, and coronary death. Cohorts conducted race-specific analyses adjusted for age, sex, smoking, education, body mass index, blood cell type proportions, and technical variables. We conducted fixed-effect meta-analyses across cohorts. RESULTS: Among 11 461 individuals (mean age 64 years, 67% women, 35% African American) free of CHD at baseline, 1895 developed CHD during a mean follow-up of 11.2 years. Methylation levels at 52 CpG (cytosine-phosphate-guanine) sites were associated with incident CHD or myocardial infarction (false discovery rate<0.05). These CpGs map to genes with key roles in calcium regulation (ATP2B2, CASR, GUCA1B, HPCAL1), and genes identified in genome- and epigenome-wide studies of serum calcium (CASR), serum calcium-related risk of CHD (CASR), coronary artery calcified plaque (PTPRN2), and kidney function (CDH23, HPCAL1), among others. Mendelian randomization analyses supported a causal effect of DNA methylation on incident CHD; these CpGs map to active regulatory regions proximal to long non-coding RNA transcripts. CONCLUSION: Methylation of blood-derived DNA is associated with risk of future CHD across diverse populations and may serve as an informative tool for gaining further insight on the development of CHD.

Association of Methylation Signals With Incident Coronary Heart Disease in an Epigenome-Wide Assessment of Circulating Tumor Necrosis Factor α.

Importance: Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine with manifold consequences for mammalian pathophysiology, including cardiovascular disease. A deeper understanding of TNF-α biology may enhance treatment precision. Objective: To conduct an epigenome-wide analysis of blood-derived DNA methylation and TNF-α levels and to assess the clinical relevance of findings. Design, Setting, and Participants: This meta-analysis assessed epigenome-wide associations in circulating TNF-α concentrations from 5 cohort studies and 1 interventional trial, with replication in 3 additional cohort studies. Follow-up analyses investigated associations of identified methylation loci with gene expression and incident coronary heart disease; this meta-analysis included 11 461 participants who experienced 1895 coronary events. Exposures: Circulating TNF-α concentration. Main Outcomes and Measures: DNA methylation at approximately 450 000 loci, neighboring DNA sequence variation, gene expression, and incident coronary heart disease. Results: The discovery cohort included 4794 participants, and the replication study included 816 participants (overall mean [SD] age, 60.7 [8.5] years). In the discovery stage, circulating TNF-α levels were associated with methylation of 7 cytosine-phosphate-guanine (CpG) sites, 3 of which were located in or near DTX3L-PARP9 at cg00959259 (ß [SE] = -0.01 [0.003]; P = 7.36 × 10-8), cg08122652 (ß [SE] = -0.008 [0.002]; P = 2.24 × 10-7), and cg22930808(ß [SE] = -0.01 [0.002]; P = 6.92 × 10-8); NLRC5 at cg16411857 (ß [SE] = -0.01 [0.002]; P = 2.14 × 10-13) and cg07839457 (ß [SE] = -0.02 [0.003]; P = 6.31 × 10-10); or ABO, at cg13683939 (ß [SE] = 0.04 [0.008]; P = 1.42 × 10-7) and cg24267699 (ß [SE] = -0.009 [0.002]; P = 1.67 × 10-7), after accounting for multiple testing. Of these, negative associations between TNF-α concentration and methylation of 2 loci in NLRC5 and 1 in DTX3L-14 PARP9 were replicated. Replicated TNF-α-linked CpG sites were associated with 9% to 19% decreased risk of incident coronary heart disease per 10% higher methylation per CpG site (cg16411857: hazard ratio [HR], 0.86; 95% CI, 0.78-1.95; P = .003; cg07839457: HR, 0.89; 95% CI, 0.80-0.94; P = 3.1 × 10-5; cg00959259: HR, 0.91; 95% CI, 0.84-0.97; P = .002; cg08122652: HR, 0.81; 95% CI, 0.74-0.89; P = 2.0 × 10-5). Conclusions and Relevance: We identified and replicated novel epigenetic correlates of circulating TNF-α concentration in blood samples and linked these loci to coronary heart disease risk, opening opportunities for validation and therapeutic applications.

Serum magnesium and the risk of prediabetes: a population-based cohort study.

AIMS/HYPOTHESIS: Previous studies have found an association between serum magnesium and incident diabetes; however, this association may be due to reverse causation, whereby diabetes may induce urinary magnesium loss. In contrast, in prediabetes (defined as impaired fasting glucose), serum glucose levels are below the threshold for urinary magnesium wasting and, hence, unlikely to influence serum magnesium levels. Thus, to study the directionality of the association between serum magnesium levels and diabetes, we investigated its association with prediabetes. We also investigated whether magnesium-regulating genes influence diabetes risk through serum magnesium levels. Additionally, we quantified the effect of insulin resistance in the association between serum magnesium levels and diabetes risk. METHODS: Within the population-based Rotterdam Study, we used Cox models, adjusted for age, sex, lifestyle factors, comorbidities, kidney function, serum levels of electrolytes and diuretic use, to study the association between serum magnesium and prediabetes/diabetes. In addition, we performed two mediation analyses: (1) to study if common genetic variation in eight magnesium-regulating genes influence diabetes risk through serum magnesium levels; and (2) to quantify the proportion of the effect of serum magnesium levels on diabetes that is mediated through insulin resistance (quantified by HOMA-IR). RESULTS: A total of 8555 participants (mean age, 64.7 years; median follow-up, 5.7 years) with normal glucose levels (mean ± SD: 5.46 ± 0.58 mmol/l) at baseline were included. A 0.1 mmol/l decrease in serum magnesium level was associated with an increase in diabetes risk (HR 1.18 [95% CI 1.04, 1.33]), confirming findings from previous studies. Of interest, a similar association was found between serum magnesium levels and prediabetes risk (HR 1.12 [95% CI 1.01, 1.25]). Genetic variation in CLDN19, CNNM2, FXYD2, SLC41A2, and TRPM6 significantly influenced diabetes risk (p < 0.05), and for CNNM2, FXYD2, SLC41A2 and TRPM6 this risk was completely mediated by serum magnesium levels. We found that 29.1% of the effect of serum magnesium levels on diabetes was mediated through insulin resistance, whereas for prediabetes 13.4% was mediated through insulin resistance. CONCLUSIONS/INTERPRETATION: Low serum magnesium levels are associated with an increased risk of prediabetes and this increased risk is similar to that of diabetes. Furthermore, common variants in magnesium-regulating genes modify diabetes risk through serum magnesium levels. Both findings support a potential causal role of magnesium in the development of diabetes, where the hypothesised pathway is partly mediated through insulin resistance.

Serum Levels of Apolipoproteins and Incident Type 2 Diabetes: A Prospective Cohort Study.

OBJECTIVE: We aimed to investigate the role of serum levels of various apolipoproteins on the risk for type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: We used data from 971 individuals from the prospective population-based Rotterdam Study. We studied the association of HDL cholesterol (HDL-C), apoA1, apoCIII, apoD, and apoE as well as the ratios of apolipoproteins with apoA1 with the risk of T2D. All apolipoproteins, ratios, and HDL-C levels were naturally log-transformed to reach normal distribution. First, their cross-sectional associations with fasting glucose and insulin were investigated by using linear regression. Second, Cox proportional hazard models were used to examine whether apolipoproteins predict the risk for T2D among individuals free of diabetes at baseline. We also studied the apolipoproteins jointly by calculating the apolipoproteinic score from the first step and then performing Cox regression with it. RESULTS: During a median follow-up of 13.5 years, diabetes developed in 110 individuals. After adjustment for age, sex, BMI, parental history of diabetes, hypertension, alcohol use, smoking, prevalent cardiovascular disease, and serum lipid-reducing agents, HDL-C (per 1 SD naturally log-transformed hazard ratio 0.74 [95% CI 0.57, 0.97], apoCIII (1.65 [1.42, 1.91]), apoE (1.36 [1.18, 1.55]), apoCIII-to-apoA1 ratio (1.72 [1.51, 1.95]), apoE-to-apoA1 ratio (1.28 [1.13, 1.45]), and apolipoproteinic score (1.60 [1.39, 1.83]) remained significant. Only apoCIII (1.42 [1.03, 1.96]) and apoCIII-to-apoA1 ratio (1.56 [1.04, 2.36]) survived the adjustment for triglycerides in the last model. CONCLUSIONS: Serum apoCIII levels as well as apoCIII-to-apoA1 ratio are associated with incident T2D. They are associated independent of known risk factors and stronger than HDL-C levels.

Metabolic syndrome is related to polyneuropathy and impaired peripheral nerve function: a prospective population-based cohort study.

OBJECTIVE: Diabetes mellitus is a known risk factor for polyneuropathy, but the role of pre-diabetes and metabolic syndrome remains unclear. We aimed to investigate the role of these factors in a community-dwelling middle-aged and elderly population. METHODS: 1256 participants of the population-based Rotterdam Study (mean age 70.0, 54.5% females) were screened for polyneuropathy with a questionnaire, neurological examination and nerve conduction studies. Data on type 2 diabetes and components of metabolic syndrome were also collected. Logistic regression was used to investigate associations of diabetes, pre-diabetes and metabolic syndrome and its separate components with polyneuropathy. Linear regression was used to investigate associations with nerve conduction parameters in participants without polyneuropathy. FINDINGS: Diabetes was associated with polyneuropathy (OR 3.01, 95% CI 1.60 to 5.65), while impaired fasting glucose was not (OR 1.55, 95% CI 0.70 to 3.44). Metabolic syndrome was associated with polyneuropathy (OR 1.92, 95% CI 1.09 to 3.38), with a stronger association when more components of the syndrome were present. Analysing separate components of metabolic syndrome revealed associations for elevated waist circumference (OR 2.84, 95% CI 1.35 to 5.99) and elevated triglycerides (OR 2.01, 95% CI 1.11 to 3.62). Similar associations were found after excluding participants with diabetes. In participants without polyneuropathy, metabolic syndrome associated with lower sural sensory nerve action potential amplitudes. CONCLUSIONS: Metabolic syndrome, abdominal obesity and dyslipidaemia, are strongly associated with polyneuropathy, irrespective of the presence of diabetes. Metabolic syndrome also associates with impaired nerve function in people without polyneuropathy. Our study therefore suggests that cardiometabolic disturbances have an impact on peripheral nerve function that extends beyond clinically manifest disease.

DNA Methylation in Newborns and Maternal Smoking in Pregnancy: Genome-wide Consortium Meta-analysis.

Epigenetic modifications, including DNA methylation, represent a potential mechanism for environmental impacts on human disease. Maternal smoking in pregnancy remains an important public health problem that impacts child health in a myriad of ways and has potential lifelong consequences. The mechanisms are largely unknown, but epigenetics most likely plays a role. We formed the Pregnancy And Childhood Epigenetics (PACE) consortium and meta-analyzed, across 13 cohorts (n = 6,685), the association between maternal smoking in pregnancy and newborn blood DNA methylation at over 450,000 CpG sites (CpGs) by using the Illumina 450K BeadChip. Over 6,000 CpGs were differentially methylated in relation to maternal smoking at genome-wide statistical significance (false discovery rate, 5%), including 2,965 CpGs corresponding to 2,017 genes not previously related to smoking and methylation in either newborns or adults. Several genes are relevant to diseases that can be caused by maternal smoking (e.g., orofacial clefts and asthma) or adult smoking (e.g., certain cancers). A number of differentially methylated CpGs were associated with gene expression. We observed enrichment in pathways and processes critical to development. In older children (5 cohorts, n = 3,187), 100% of CpGs gave at least nominal levels of significance, far more than expected by chance (p value < 2.2 × 10(-16)). Results were robust to different normalization methods used across studies and cell type adjustment. In this large scale meta-analysis of methylation data, we identified numerous loci involved in response to maternal smoking in pregnancy with persistence into later childhood and provide insights into mechanisms underlying effects of this important exposure.

Tobacco smoking is associated with DNA methylation of diabetes susceptibility genes.

AIMS/HYPOTHESIS: Tobacco smoking, a risk factor for diabetes, is an established modifier of DNA methylation. We hypothesised that tobacco smoking modifies DNA methylation of genes previously identified for diabetes. METHODS: We annotated CpG sites available on the Illumina Human Methylation 450K array to diabetes genes previously identified by genome-wide association studies (GWAS), and investigated them for an association with smoking by comparing current to never smokers. The discovery study consisted of 630 individuals (Bonferroni-corrected p = 1.4 × 10(-5)), and we sought replication in an independent sample of 674 individuals. The replicated sites were tested for association with nearby genetic variants and gene expression and fasting glucose and insulin levels. RESULTS: We annotated 3,620 CpG sites to the genes identified in the GWAS on type 2 diabetes. Comparing current smokers to never smokers, we found 12 differentially methylated CpG sites, of which five replicated: cg23161492 within ANPEP (p = 1.3 × 10(-12)); cg26963277 (p = 1.2 × 10(-9)), cg01744331 (p = 8.0 × 10(-6)) and cg16556677 (p = 1.2 × 10(-5)) within KCNQ1 and cg03450842 (p = 3.1 × 10(-8)) within ZMIZ1. The effect of smoking on DNA methylation at the replicated CpG sites attenuated after smoking cessation. Increased DNA methylation at cg23161492 was associated with decreased gene expression levels of ANPEP (p = 8.9 × 10(-5)). rs231356-T, which was associated with hypomethylation of cg26963277 (KCNQ1), was associated with a higher odds of diabetes (OR 1.06, p = 1.3 × 10(-5)). Additionally, hypomethylation of cg26963277 was associated with lower fasting insulin levels (p = 0.04). CONCLUSIONS/INTERPRETATION: Tobacco smoking is associated with differential DNA methylation of the diabetes risk genes ANPEP, KCNQ1 and ZMIZ1. Our study highlights potential biological mechanisms connecting tobacco smoking to excess risk of type 2 diabetes.

Tobacco smoking is associated with methylation of genes related to coronary artery disease.

BACKGROUND: Tobacco smoking, a risk factor for coronary artery disease (CAD), is known to modify DNA methylation. We hypothesized that tobacco smoking modifies methylation of the genes identified for CAD by genome-wide association study (GWAS). RESULTS: We selected genomic regions based on 150 single-nucleotide polymorphisms (SNPs) identified in the largest GWAS on CAD. We investigated the association between current smoking and the CpG sites within and near these CAD-related genes. Methylation was measured with the Illumina Human Methylation 450K array in whole blood of 724 Caucasian subjects from the Rotterdam Study, a Dutch population based cohort study. A total of 3669 CpG sites within 169 CAD-related genes were studied for association with current compared to never smoking. Fifteen CpG sites were significantly associated after correction for multiple testing (Bonferroni-corrected p value <1.4 × 10(-5)). These sites were located in the genes TERT, SARS, GNGT2, SMG6, SKI, TOM1L2, SIPA1, MRAS, CDKN1A, LRRC2, FES and RPH3A. In 12 sites, current smoking was associated with a 1.2 to 2.4 % lower methylation compared to never smoking; and in three sites, it was associated with a 1.2 to 1.8 % higher methylation. The effect estimates were lower in 10 of the 15 CpG sites when comparing current to former smoking. One CpG site, cg05603985 (SKI), was found to be associated with expression of nearby CAD-related gene PRKCZ. CONCLUSIONS: Our study suggests an effect of tobacco smoking on DNA methylation of CAD-related genes and thus provides novel insights in the pathways that link tobacco smoking to risk of CAD.