Identified <i>cis</i>-mQTLs/eQTLs where methylation of CpG sites is a potential mediator of genetic association with mRNA expression in human pancreatic islets.

<p>Causal Inference Test (CIT) was used to test if associations between SNPs identified in the <i>cis</i>-mQTL analysis and islet mRNA expression was mediated by DNA methylation of CpG sites.</p><p>G vs M: Adjusted associations between genotype (G) and DNA methylation (M) – <i>cis</i>-mQTL analysis. P-values corrected for multiple testing based on the number of independent tests performed.</p><p>G vs E: Adjusted associations between genotype (G) and human islet mRNA expression (E) – eQTL analysis of <i>cis</i>-mQTL-SNPs. P-values corrected for multiple testing based on the number of independent tests performed.</p><p>CIT: Causal hypothesis at 5% FDR (Q-value<0.05) showing that the relationship between genotype (G) and islet mRNA expression (E) is potentially mediated through DNA methylation (M).</p><p>Identified <i>cis</i>-mQTLs/eQTLs where methylation of CpG sites is a potential mediator of genetic association with mRNA expression in human pancreatic islets.</p

CIT analysis identifies mQTLs where DNA methylation potentially mediates genetic associations with mRNA expression in human pancreatic islets.

<p>(<b>A</b>) Depiction of possible relationship models between genotype as a causal factor (G), DNA methylation as a potential mediator (M) and islet mRNA expression as a phenotypic outcome (E). Left diagram: The causal or methylation mediated model. Middle diagram: The reactive or methylation-consequential model (reverse causality). Right diagram: The independent model. (<b>B</b>) Illustration of the study approach to identify if DNA methylation of CpG sites potentially mediates the causal association between SNPs and islet mRNA expression. Left: Workflow steps. Middle: Tested relationships between G, M and E in the different steps. Right: Number of identified sites in each step. Bottom: Conditions that must be fulfilled to conclude a mathematical definition of a causal relationship between G, M and E. Significantly called as causal at 5% FDR (causal hypothesis Q<0.05).</p

Number of significant eQTL results in the human pancreatic islets.

<p>Only SNPs of significant mQTLs are included in the eQTL analysis.</p><p>SNPs of significant <i>cis</i>-mQTLs are regressed against mRNA expression of transcripts located in <i>cis</i> (≤500 kb).</p><p>SNPs of significant <i>trans</i>-mQTLs are regressed against mRNA expression of all transcripts.</p><p>Significance threshold <0.05 after correction for multiple testing.</p><p>Correction value of eQTL analysis for <i>cis</i>-mQTL-SNPs = 692, 616.</p><p>Correction value of eQTL analysis for <i>trans</i>-mQTL-SNPs = 16,982,420.</p><p>LD = linkage disequilibrium.</p><p>Number of significant eQTL results in the human pancreatic islets.</p

Identified mQTL/eQTL candidate genes <i>GPX7</i>, <i>GSTT1</i> and <i>SNX19</i> affect β-cell number and apoptosis.

<p>Associations identified in the mQTL/eQTL analyses of human pancreatic islets. (<b>A</b>) rs835342 located approximately 5 kb upstream of <i>GPX7</i> associates with DNA methylation of cg18087326 located 406 bp upstream of the <i>GPX7</i> transcription start site (TSS) as well as with mRNA expression of <i>GPX7</i>. (<b>B</b>) rs4822453 located ∼121 kb downstream of <i>GSTT1</i> associates with DNA methylation of cg17005068 located 241 bp upstream of the <i>GSTT1</i> TSS as well as with mRNA expression of <i>GSTT1</i>. (<b>C</b>) rs3751035 located within exon 1 of <i>SNX19</i> associates with DNA methylation of cg08912652 located within the gene body of <i>SNX19</i> as well as with mRNA expression of <i>SNX19</i>. Data are presented as Box and Whisker plots with P-values adjusted for multiple testing. (<b>D</b>) qPCR quantification of siRNA mediated knockdown of <i>Gpx7</i> (siGpx7), <i>Gstt1</i> (siGstt1) and <i>Snx19</i> (siSnx19) compared to negative control siRNA (siNC). * P<0.01, the graphs show the average of four independent knockdown experiments presented as mean ± SEM. (<b>E</b>) Knockdown of <i>Gpx7</i> and <i>Gstt1</i> resulted in increased combined caspase-3/7 activity compared to negative control siRNA under both control (white bars) and lipotoxic (black bars) conditions. * P<0.05, the graph shows the average of three independent knockdown experiments presented as mean ± SEM. (<b>F</b>) Knockdown of <i>Snx19</i> (siSnx19) resulted in increased cell number compared to negative control siRNA (siNC) under both control (white bars) and lipotoxic (black bars) conditions. * P<0.05, the graph shows the average of six independent knockdown experiments presented as mean ± SEM.</p

Genomic distribution of CpG sites of significantly identified mQTLs in human pancreatic islets.

<p>(<b>A</b>) Chromosomal distribution of CpG sites of significant <i>cis</i>- and <i>trans</i>-mQTLs in comparison to all analyzed CpG sites on the Infinium Human Methylation450 BeadChip. (<b>B</b>) All analyzed CpG sites on the Infinium Human Methylation450 BeadChip have been annotated to genomic regions based on their relation to the nearest gene (TSS: proximal promoter, defined as 200 bp or 1500 bp upstream of transcription start site; UTR: untranslated region) or in relation to the nearest CpG island (CpG island: DNA stretch of 200 bp or more with a C+G content of >50% and an observed CpG/expected CpG in excess of 0.6; Shore: the flanking region of CpG islands, 0–2000 bp; Shelf: regions flanking island shores, i.e., covering 2000–4000 bp distant from the CpG island). Distribution of CpG sites of significant mQTLs in relation to (<b>C</b>) the nearest gene and (<b>D</b>) in relation to CpG islands. *Significantly different distribution (P<0.05) of CpGs of significant <i>cis</i>- or <i>trans</i>-mQTLs from what is expected by chance based on a Chi-squared-test when compared with all analyzed CpG sites on the Infinium HumanMethylation450 BeadChip.</p

Flow-chart showing the analysis pipeline.

<p>Direction of the arrows represents the workflow of the study design with performed analysis indicated. Solid lines indicate analysis performed within data of human pancreatic islets. Dashed lines indicate analysis performed against external databases. Light grey boxes indicate input data of human pancreatic islets. Dark grey boxes indicate output of significant data. White boxes indicate follow-up studies for look-up or functional- and biological validation of significant results.</p

mQTLs/eQTLs of <i>GPX7</i> and <i>SNX19</i> identified in the genome-wide analysis were biologically validated in pancreatic islets from a different set of human donors.

<p>Biological validation of associations for (<b>A</b>) <i>GPX7</i> rs835342 with DNA methylation of cg18087326 as well as with mRNA expression of <i>GPX7</i> and (<b>B</b>) <i>SNX19</i> rs3751035 with DNA methylation of cg08912652 as well as with mRNA expression of <i>SNX19</i> in a set of human pancreatic islets from donors (n = 37) not included in the genome-wide mQTL/eQTL analysis. DNA methylation was analyzed using Pyrosequencing and mRNA expression using Affymetrix microarray. Data are presented as Box and Whisker plots with P-values.</p

Number of significant mQTL results in human pancreatic islets when including imputed genotyped data.

<p>Significance threshold <0.05 after correction for multiple testing.</p><p>Correction value <i>cis</i> = 102,307,720.</p><p>Correction value <i>trans</i> = 200,388,516,440.</p><p>Number of significant mQTL results in human pancreatic islets when including imputed genotyped data.</p