Abstract
Aims/hypothesisTobacco 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.MethodsWe 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.ResultsWe 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/interpretationTobacco 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3872-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Highlights
In the last decade, genome-wide association studies (GWAS) have been conducted in order to identify DNA sequence variants for a wide range of diseases including type 2 diabetes [1,2,3]
Clinical characteristics of the replication population can be found in electronic supplementary material (ESM) Table 1
Our findings suggest that tobacco smoking is associated with differential methylation of CpG sites within the type 2 diabetes risk genes ANPEP, KCNQ1 and ZMIZ1
Summary
Study population The study was conducted using data from the Rotterdam Study; the design of the Rotterdam Study has been described elsewhere [12]. Genes were declared significantly expressed when the detection p values calculated by GenomeStudio were less than 0.05 in more than 10% of all discovery samples, which added to a total number of 21,238 probes. The 3,620 methylation probes were tested for association with tobacco smoking using a linear mixed model with the LME4 package in R version 3.1.0 with Dasen-normalised β values of the CpG sites as outcome measure (https://cran.r-project.org/ web/packages/lme4/index.html) [20]. We created residuals for DNA methylation regressing out the measured white blood cells, age, sex, array number and position on array on the Dasen-normalised β values of the CpG sites using a linear mixed model. The residuals of the mRNA expression levels and the residuals of the Dasen-normalised β values of the CpG sites were tested for association using a linear regression model. Significant met-QTLs were tested for an association with type 2 diabetes in the publicly available data from the DIAGRAM consortium, using a Bonferronicorrected p value of 0.01 (0.05 / 5 met-QTLs) [3]
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