DNA methylation profiles in type 1 diabetes twins point to strong epigenetic effects on etiology

Abstract

Type 1 diabetes (T1D) shows ∼40% concordance rate in monozygotic twins (MZ) suggesting a role for environmental factors and/or epigenetic modifications in the etiology of the disease. The aim of our study was to dissect the contribution of epigenetic factors, particularly, DNA methylation (DNAm), to the incomplete penetrance of T1D. We performed DNAm profiling in lymphocyte cell lines from 3 monozygotic (MZ) twin pairs discordant for T1D and 6 MZ twin pairs concordant for the disease using HumanMethylation27 BeadChip. This assay assesses the methylation state of 27,578 CpG sites, mostly located within proximal promoter regions. We identified 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. Functional annotation of the genes with distinct CpG methylation profiles in T1D samples showed differential DNAm of immune response and defense response pathways between affected and unaffected twins. Integration of DNAm data with GWAS data mapped several known T1D associated genes, HLA, INS, IL-2RB, CD226, which showed significant differences in DNAm between affected and unaffected of twins. Our findings suggest that abnormalities of DNA methylation patterns, known to regulate gene transcription, may be involved in the pathogenesis of T1D.

Keywords: DNA methylation; Monozygotic twins; Transcriptional regulation; Type 1 diabetes.

Figure 1

(A) Pie chart showing the 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. More CpG sites were hyper-methylated (55 sites) compared with hypo-methylated (33 sites); (B) Distribution of hyper- and hypo-methylated CpG islands (CGI) across four annotation sets relative to the TSS (± 1 kb from TSS) in discordant twins. TSS-CGI = CGIs present 1Kb up- and down-stream the TSS; TSS-non CGI = no CGIs present 1Kb up- and down-stream the TSS; nonTSS-nonCGI = regions outside TSS with no CGIs; nonTSS-CGI = CGIs present outside TSS ± 1Kb; (C) Heatmap of differentially methylated CpG loci showing different methylation patterns in affected vs. unaffected individuals for both discordant (yellow line) and concordant (blue line) MZ twin pairs; Median methylation level of unaffected twins was used as baseline. 1.0 and −1.0 represents 2-fold increase or decrease methylation respectively compared to the baseline. A, affected twin; U, unaffected twin; CT, concordant MZ twin pairs; DT, discordant MZ twin pairs; (D) DNA methylation levels in the CCDC3 gene: 20 CpGs from promoter CGI were analyzed in 8 twin pairs. All samples showed reduced DNA methylation; the levels of methylation in the cg20647888 array probe did not differ from the overall level of the entire CGI. Insert: an example of bisulfite sequencing of multiple clones from the DT2 twin pair. Schematic representation of the methylation status of each CpG. Black and white circles indicate methylated and unmethyalted CpGs, respectively. Horizontal line indicates methylation sites at the 4 CpGs present on cg20647888. (E) Confirmation of the DNAm levels in the Kv2.2 gene in all twin pairs by direct sequencing of bisulfite modified DNA. 10 CpGs form Kv2.2 CGI were analyzed in all twin pairs. With exception of one CT pair (CT1) all affected individuals had increased CpG methylation within Kv2.2 promoter; U = unaffected twin, A = affected twin.

Figure 1

(A) Pie chart showing the 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. More CpG sites were hyper-methylated (55 sites) compared with hypo-methylated (33 sites); (B) Distribution of hyper- and hypo-methylated CpG islands (CGI) across four annotation sets relative to the TSS (± 1 kb from TSS) in discordant twins. TSS-CGI = CGIs present 1Kb up- and down-stream the TSS; TSS-non CGI = no CGIs present 1Kb up- and down-stream the TSS; nonTSS-nonCGI = regions outside TSS with no CGIs; nonTSS-CGI = CGIs present outside TSS ± 1Kb; (C) Heatmap of differentially methylated CpG loci showing different methylation patterns in affected vs. unaffected individuals for both discordant (yellow line) and concordant (blue line) MZ twin pairs; Median methylation level of unaffected twins was used as baseline. 1.0 and −1.0 represents 2-fold increase or decrease methylation respectively compared to the baseline. A, affected twin; U, unaffected twin; CT, concordant MZ twin pairs; DT, discordant MZ twin pairs; (D) DNA methylation levels in the CCDC3 gene: 20 CpGs from promoter CGI were analyzed in 8 twin pairs. All samples showed reduced DNA methylation; the levels of methylation in the cg20647888 array probe did not differ from the overall level of the entire CGI. Insert: an example of bisulfite sequencing of multiple clones from the DT2 twin pair. Schematic representation of the methylation status of each CpG. Black and white circles indicate methylated and unmethyalted CpGs, respectively. Horizontal line indicates methylation sites at the 4 CpGs present on cg20647888. (E) Confirmation of the DNAm levels in the Kv2.2 gene in all twin pairs by direct sequencing of bisulfite modified DNA. 10 CpGs form Kv2.2 CGI were analyzed in all twin pairs. With exception of one CT pair (CT1) all affected individuals had increased CpG methylation within Kv2.2 promoter; U = unaffected twin, A = affected twin.

Figure 1

(A) Pie chart showing the 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. More CpG sites were hyper-methylated (55 sites) compared with hypo-methylated (33 sites); (B) Distribution of hyper- and hypo-methylated CpG islands (CGI) across four annotation sets relative to the TSS (± 1 kb from TSS) in discordant twins. TSS-CGI = CGIs present 1Kb up- and down-stream the TSS; TSS-non CGI = no CGIs present 1Kb up- and down-stream the TSS; nonTSS-nonCGI = regions outside TSS with no CGIs; nonTSS-CGI = CGIs present outside TSS ± 1Kb; (C) Heatmap of differentially methylated CpG loci showing different methylation patterns in affected vs. unaffected individuals for both discordant (yellow line) and concordant (blue line) MZ twin pairs; Median methylation level of unaffected twins was used as baseline. 1.0 and −1.0 represents 2-fold increase or decrease methylation respectively compared to the baseline. A, affected twin; U, unaffected twin; CT, concordant MZ twin pairs; DT, discordant MZ twin pairs; (D) DNA methylation levels in the CCDC3 gene: 20 CpGs from promoter CGI were analyzed in 8 twin pairs. All samples showed reduced DNA methylation; the levels of methylation in the cg20647888 array probe did not differ from the overall level of the entire CGI. Insert: an example of bisulfite sequencing of multiple clones from the DT2 twin pair. Schematic representation of the methylation status of each CpG. Black and white circles indicate methylated and unmethyalted CpGs, respectively. Horizontal line indicates methylation sites at the 4 CpGs present on cg20647888. (E) Confirmation of the DNAm levels in the Kv2.2 gene in all twin pairs by direct sequencing of bisulfite modified DNA. 10 CpGs form Kv2.2 CGI were analyzed in all twin pairs. With exception of one CT pair (CT1) all affected individuals had increased CpG methylation within Kv2.2 promoter; U = unaffected twin, A = affected twin.