Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes

Delphine Fradin, Sophie Le Fur, Clémence Mille, Nadia Naoui, Chris Groves, Diana Zelenika, Mark I McCarthy, Mark Lathrop, Pierre Bougnères, Delphine Fradin, Sophie Le Fur, Clémence Mille, Nadia Naoui, Chris Groves, Diana Zelenika, Mark I McCarthy, Mark Lathrop, Pierre Bougnères

Abstract

The insulin (INS) region is the second most important locus associated with Type 1 Diabetes (T1D). The study of the DNA methylation pattern of the 7 CpGs proximal to the TSS in the INS gene promoter revealed that T1D patients have a lower level of methylation of CpG -19, -135 and -234 (p = 2.10(-16)) and a higher methylation of CpG -180 than controls, while methylation was comparable for CpG -69, -102, -206. The magnitude of the hypomethylation relative to a control population was 8-15% of the corresponding levels in controls and was correlated in CpGs -19 and -135 (r = 0.77) and CpG -135 and -234 (r = 0.65). 70/485 (14%) of T1D patients had a simultaneous decrease in methylation of CpG -19, -135, -234 versus none in 317 controls. CpG methylation did not correlate with glycated hemoglobin or with T1D duration. The methylation of CpG -69, -102, -180, -206, but not CpG -19, -135, -234 was strongly influenced by the cis-genotype at rs689, a SNP known to show a strong association with T1D. We hypothesize that part of this genetic association could in fact be mediated at the statistical and functional level by the underlying changes in neighboring CpG methylation. Our observation of a CpG-specific, locus-specific methylation pattern, although it can provide an epigenetic biomarker of a multifactorial disease, does not indicate whether the reported epigenetic pattern preexists or follows the establishment of T1D. To explore the effect of chronic hyperglycemia on CpG methylation, we studied non obese patients with type 2 diabetes (T2D) who were found to have decreased CpG-19 methylation versus age-matched controls, similar to T1D (p = 2.10(-6)) but increased CpG-234 methylation (p = 5.10(-8)), the opposite of T1D. The causality and natural history of the different epigenetic changes associated with T1D or T2D remain to be determined.

Conflict of interest statement

Competing Interests: The study was partly funded by NovoNordisk. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1. Heatmap analysis of the relationship…
Figure 1. Heatmap analysis of the relationship between DNA methylation of the whole proximal INS promoter and disease status.
Each row represents one individual.
Figure 2. Correlation matrix of the methylation…
Figure 2. Correlation matrix of the methylation values (%) at the INS promoter CpG sites in T1D patients (R in bold, p-value below).
The T1D-associated hypomethylated CpGs -19, -135 and -234 are highly correlated (−16).
Figure 3. Lack of correlation between age…
Figure 3. Lack of correlation between age and INS promoter methylation in T1D patients (green) and Controls (red).
Only CpG -180 methylation showed a slight downward trend with age (r = 0.09, p = 0.01). The figure also shows the distribution of individual methylation values, and the differences between T1D and control subjects for CpGs -19, -135, -180, and -234.
Figure 4. UCSC Genome Browser map of…
Figure 4. UCSC Genome Browser map of the INS locus : localization of genes, CpG islands (CGI), variants and CpG sites.
Genes are represented by blue boxes (exons) and lines (intron). Under genes, CGI are in green. Green intensity and size of the box is function of the size and the number of CpG included in CGI. Polymorphisms are represented by black boxes, under CGI. Below, a zoom of the INS promoter allowing the localization and representation of the isolated CpG sites, according to the TSS, represented by the red arrow.
Figure 5. Boxplot of methylation values of…
Figure 5. Boxplot of methylation values of the studied CpGs in the INS promoter with respect to the rs689 genotype in T1D patients.
Except for the CpG -180, the T1D-associated CpGs show no direct association with the genotype. P-values are calculated by Kruskal-Wallis test.
Figure 6. Boxplot of methylation values for…
Figure 6. Boxplot of methylation values for the 7 CpGs in INS promoter with respect to SNPs other than rs689 in T1D patients.
Methylation at CpGs -69, −102, −180 and −206 are associated with rs3842748. More distant SNPs showed a much weaker correlation with methylation at CpG -69 and -102 for rs4320932 and with methylation at CpGs -102, -135, -180 and −206 for rs6356. See Figure S1 for SNP locations. P-values are calculated by Krukal-Wallis test.

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