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Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets.

Olsson AH, Volkov P, Bacos K, Dayeh T, Hall E, Nilsson EA, Ladenvall C, Rönn T, Ling C - PLoS Genet. (2014)

Bottom Line: CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands.Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets.Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences, Epigenetics and Diabetes, Lund University Diabetes Centre, Clinical Research Centre, Malmö, Sweden.

ABSTRACT
Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic variation interacts to influence gene expression, islet function and potential diabetes risk in humans.

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Identified mQTL/eQTL candidate genes GPX7, GSTT1 and SNX19 affect β-cell number and apoptosis.Associations identified in the mQTL/eQTL analyses of human pancreatic islets. (A) rs835342 located approximately 5 kb upstream of GPX7 associates with DNA methylation of cg18087326 located 406 bp upstream of the GPX7 transcription start site (TSS) as well as with mRNA expression of GPX7. (B) rs4822453 located ∼121 kb downstream of GSTT1 associates with DNA methylation of cg17005068 located 241 bp upstream of the GSTT1 TSS as well as with mRNA expression of GSTT1. (C) rs3751035 located within exon 1 of SNX19 associates with DNA methylation of cg08912652 located within the gene body of SNX19 as well as with mRNA expression of SNX19. Data are presented as Box and Whisker plots with P-values adjusted for multiple testing. (D) qPCR quantification of siRNA mediated knockdown of Gpx7 (siGpx7), Gstt1 (siGstt1) and Snx19 (siSnx19) compared to negative control siRNA (siNC). * P<0.01, the graphs show the average of four independent knockdown experiments presented as mean ± SEM. (E) Knockdown of Gpx7 and Gstt1 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. (F) Knockdown of Snx19 (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.
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pgen-1004735-g005: Identified mQTL/eQTL candidate genes GPX7, GSTT1 and SNX19 affect β-cell number and apoptosis.Associations identified in the mQTL/eQTL analyses of human pancreatic islets. (A) rs835342 located approximately 5 kb upstream of GPX7 associates with DNA methylation of cg18087326 located 406 bp upstream of the GPX7 transcription start site (TSS) as well as with mRNA expression of GPX7. (B) rs4822453 located ∼121 kb downstream of GSTT1 associates with DNA methylation of cg17005068 located 241 bp upstream of the GSTT1 TSS as well as with mRNA expression of GSTT1. (C) rs3751035 located within exon 1 of SNX19 associates with DNA methylation of cg08912652 located within the gene body of SNX19 as well as with mRNA expression of SNX19. Data are presented as Box and Whisker plots with P-values adjusted for multiple testing. (D) qPCR quantification of siRNA mediated knockdown of Gpx7 (siGpx7), Gstt1 (siGstt1) and Snx19 (siSnx19) compared to negative control siRNA (siNC). * P<0.01, the graphs show the average of four independent knockdown experiments presented as mean ± SEM. (E) Knockdown of Gpx7 and Gstt1 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. (F) Knockdown of Snx19 (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.

Mentions: To examine whether altered expression of some of the identified candidate genes in the islet mQTL/eQTL analyses affect β-cell function and thereby potentially the development of diabetes, we silenced the expression of three selected genes; Gpx7, Gstt1 and Snx19, in clonal β-cells. These genes were selected based on their potential role in diabetes and islet function [47], [49]–[51] and because they showed both differential DNA methylation and gene expression between genotype groups in the mQTL and eQTL analyses (Table S2 and Table S5). One representative mQTL and eQTL for GPX7,, GSTT1 and SNX19, respectively, is presented in Figure 5A–C. Moreover, GPX7 and GSTT1 belong to the genes that were enriched in the glutathione metabolism KEGG pathway of significant cis-eQTLs. The knock-down experiments were performed to establish if identified genes in our mQTL analysis have a biological function in pancreatic β-cells. While both GPX7 and GSTT1 encode proteins that are known to protect against oxidative stress [48], [52], [53], sortin nexin 19, encoded by SNX19, may put cells into a pre-apoptotic state [50]. We therefore studied cell number and cell death signaling, measured as caspase-3/7 activities, under control and lipotoxic stress conditions when silencing selected candidate genes in clonal β-cells. The expression level of Gpx7, Gstt1 and Snx19 respectively, was significantly reduced in the siRNA knockdown experiments (P<0.05, Figure 5D). Interestingly, both under control and lipotoxic conditions, we found increased caspase-3/7 activities in β-cells with silenced Gpx7 or Gstt1 expression compared to negative control siRNA transfected (siNC) β-cells (P<0.05, Figure 5E). Moreover, when crystal violet staining was used to measure β-cell number, knockdown of Snx19 resulted in increased cell number compared to negative control cells under both normal and lipotoxic conditions (P<0.05, Figure 5F).


Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets.

Olsson AH, Volkov P, Bacos K, Dayeh T, Hall E, Nilsson EA, Ladenvall C, Rönn T, Ling C - PLoS Genet. (2014)

Identified mQTL/eQTL candidate genes GPX7, GSTT1 and SNX19 affect β-cell number and apoptosis.Associations identified in the mQTL/eQTL analyses of human pancreatic islets. (A) rs835342 located approximately 5 kb upstream of GPX7 associates with DNA methylation of cg18087326 located 406 bp upstream of the GPX7 transcription start site (TSS) as well as with mRNA expression of GPX7. (B) rs4822453 located ∼121 kb downstream of GSTT1 associates with DNA methylation of cg17005068 located 241 bp upstream of the GSTT1 TSS as well as with mRNA expression of GSTT1. (C) rs3751035 located within exon 1 of SNX19 associates with DNA methylation of cg08912652 located within the gene body of SNX19 as well as with mRNA expression of SNX19. Data are presented as Box and Whisker plots with P-values adjusted for multiple testing. (D) qPCR quantification of siRNA mediated knockdown of Gpx7 (siGpx7), Gstt1 (siGstt1) and Snx19 (siSnx19) compared to negative control siRNA (siNC). * P<0.01, the graphs show the average of four independent knockdown experiments presented as mean ± SEM. (E) Knockdown of Gpx7 and Gstt1 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. (F) Knockdown of Snx19 (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.
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Related In: Results  -  Collection

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pgen-1004735-g005: Identified mQTL/eQTL candidate genes GPX7, GSTT1 and SNX19 affect β-cell number and apoptosis.Associations identified in the mQTL/eQTL analyses of human pancreatic islets. (A) rs835342 located approximately 5 kb upstream of GPX7 associates with DNA methylation of cg18087326 located 406 bp upstream of the GPX7 transcription start site (TSS) as well as with mRNA expression of GPX7. (B) rs4822453 located ∼121 kb downstream of GSTT1 associates with DNA methylation of cg17005068 located 241 bp upstream of the GSTT1 TSS as well as with mRNA expression of GSTT1. (C) rs3751035 located within exon 1 of SNX19 associates with DNA methylation of cg08912652 located within the gene body of SNX19 as well as with mRNA expression of SNX19. Data are presented as Box and Whisker plots with P-values adjusted for multiple testing. (D) qPCR quantification of siRNA mediated knockdown of Gpx7 (siGpx7), Gstt1 (siGstt1) and Snx19 (siSnx19) compared to negative control siRNA (siNC). * P<0.01, the graphs show the average of four independent knockdown experiments presented as mean ± SEM. (E) Knockdown of Gpx7 and Gstt1 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. (F) Knockdown of Snx19 (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.
Mentions: To examine whether altered expression of some of the identified candidate genes in the islet mQTL/eQTL analyses affect β-cell function and thereby potentially the development of diabetes, we silenced the expression of three selected genes; Gpx7, Gstt1 and Snx19, in clonal β-cells. These genes were selected based on their potential role in diabetes and islet function [47], [49]–[51] and because they showed both differential DNA methylation and gene expression between genotype groups in the mQTL and eQTL analyses (Table S2 and Table S5). One representative mQTL and eQTL for GPX7,, GSTT1 and SNX19, respectively, is presented in Figure 5A–C. Moreover, GPX7 and GSTT1 belong to the genes that were enriched in the glutathione metabolism KEGG pathway of significant cis-eQTLs. The knock-down experiments were performed to establish if identified genes in our mQTL analysis have a biological function in pancreatic β-cells. While both GPX7 and GSTT1 encode proteins that are known to protect against oxidative stress [48], [52], [53], sortin nexin 19, encoded by SNX19, may put cells into a pre-apoptotic state [50]. We therefore studied cell number and cell death signaling, measured as caspase-3/7 activities, under control and lipotoxic stress conditions when silencing selected candidate genes in clonal β-cells. The expression level of Gpx7, Gstt1 and Snx19 respectively, was significantly reduced in the siRNA knockdown experiments (P<0.05, Figure 5D). Interestingly, both under control and lipotoxic conditions, we found increased caspase-3/7 activities in β-cells with silenced Gpx7 or Gstt1 expression compared to negative control siRNA transfected (siNC) β-cells (P<0.05, Figure 5E). Moreover, when crystal violet staining was used to measure β-cell number, knockdown of Snx19 resulted in increased cell number compared to negative control cells under both normal and lipotoxic conditions (P<0.05, Figure 5F).

Bottom Line: CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands.Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets.Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences, Epigenetics and Diabetes, Lund University Diabetes Centre, Clinical Research Centre, Malmö, Sweden.

ABSTRACT
Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic variation interacts to influence gene expression, islet function and potential diabetes risk in humans.

Show MeSH
Related in: MedlinePlus