Longitudinal changes in DNA methylation during the onset of islet autoimmunity differentiate between reversion versus progression of islet autoimmunity

Abstract

Background: Type 1 diabetes (T1D) is preceded by a heterogenous pre-clinical phase, islet autoimmunity (IA). We aimed to identify pre vs. post-IA seroconversion (SV) changes in DNAm that differed across three IA progression phenotypes, those who lose autoantibodies (reverters), progress to clinical T1D (progressors), or maintain autoantibody levels (maintainers).

Methods: This epigenome-wide association study (EWAS) included longitudinal DNAm measurements in blood (Illumina 450K and EPIC) from participants in Diabetes Autoimmunity Study in the Young (DAISY) who developed IA, one or more islet autoantibodies on at least two consecutive visits. We compared reverters - individuals who sero-reverted, negative for all autoantibodies on at least two consecutive visits and did not develop T1D (n=41); maintainers - continued to test positive for autoantibodies but did not develop T1D (n=60); progressors - developed clinical T1D (n=42). DNAm data were measured before (pre-SV visit) and after IA (post-SV visit). Linear mixed models were used to test for differences in pre- vs post-SV changes in DNAm across the three groups. Linear mixed models were also used to test for group differences in average DNAm. Cell proportions, age, and sex were adjusted for in all models. Median follow-up across all participants was 15.5 yrs. (interquartile range (IQR): 10.8-18.7).

Results: The median age at the pre-SV visit was 2.2 yrs. (IQR: 0.8-5.3) in progressors, compared to 6.0 yrs. (IQR: 1.3-8.4) in reverters, and 5.7 yrs. (IQR: 1.4-9.7) in maintainers. Median time between the visits was similar in reverters 1.4 yrs. (IQR: 1-1.9), maintainers 1.3 yrs. (IQR: 1.0-2.0), and progressors 1.8 yrs. (IQR: 1.0-2.0). Changes in DNAm, pre- vs post-SV, differed across the groups at one site (cg16066195) and 11 regions. Average DNAm (mean of pre- and post-SV) differed across 22 regions.

Conclusion: Differentially changing DNAm regions were located in genomic areas related to beta cell function, immune cell differentiation, and immune cell function.

Keywords: DAISY; DNA methylation; islet autoimmunity; reversion; type 1 diabetes (T1D).

Figure 1

Summary of methods used to identify and prioritize DNAm candidates. Description: We used an epigenome wide association study design to identify differentially methylated positions (DMP) associated with the three islet autoimmunity progression phenotypes, reverters, maintainer, or progressors. We used two DMP models (1) an interaction model that tested whether changes in DNA methylation (DNAm) levels at single CpGs pre-IA versus post-IA differed across groups and (2) a group effect model that tested whether average methylation levels (pre- and post-IA) differed across groups. We also performed regional analyses (differentially methylated regions or DMRs) based on single CpG sites from the two models to identify regions with consistent methylation effects. We identified regions where average regional methylation levels differed between groups (μDMRs) as well as regions where changes in regional methylation levels pre- vs post-IA differed across groups (ΔDMRs). In order to prioritize regions, we tested whether the DNAm candidates identified in our analysis were associated with gene expression levels post-SV, an expression quantitative trait methylation analysis (eQTM). To account for the multiple CpGs within each DMR, we used a principal component analysis to capture common patterns across all CpGs included in the DMR. We identified cis-eQTMs (midpoint of region +/- 500 KB of the TSS of the gene) by testing the correlation between gene expression and the 1st principal component. We also tested the correlation between DNAm candidates and metabolite levels obtained from overlapping samples, a metabolite quantitative trait methylation analysis (metQTM). We used a principal component analysis to capture common patterns across all CpGs included in the candidate DMRs. We tested the correlation between metabolite levels and the 1st principal component. CpGs are represented by lollipop plots in the figure.

Figure 2

Changes in DNAm between the pre- and post-SV visits at cg16066195 across the three IA progression phenotypes. Description: (A) provides the average methylation M-values and corresponding 95% confidence intervals within the three IA progression phenotypes pre- and post-SV. (B) describes the individual level changes in methylation m-values (y-axis) between the post-SV visit relative to the pre-SV visit in the three IA progression phenotypes (x-axis). Positive values represent increasing DNAm whereas negative values represent decreasing methylation between visits. All DNAm values in (A, B) have been adjusted for age, sex, and cell proportions.

Figure 3

Differentially changing methylation region on chromosome 20 where changes in DNAm (pre- vs post-SV) differed across the three IA progression phenotypes. Description. Region on chromosome 20 loc 57426538 to 57427974 (ΔDMR1) where the change in DNA methylation (DNAm) post- vs pre-SV differed across groups. In the top panel, each dot represents the within group slopes (y-axis) or changes in DNAm m-values between the post-SV and pre-SV visit at each of the CpG sites included ΔDMR 1. The x-axis represents the position (mb) of the CpGs within the region. All slope values were adjusted for age, sex, and cell proportions. Positive values indicate methylation values increased following IA seroconversion whereas negative values indicate methylation decreased following IA seroconversion. The dashed lines represent the average slope value within each group across the entire region. The middle panel represents the location of the region (black solid square) relative to the closest genes, GNAS and ATP5E (red solid boxes). There are multiple known isoforms for GNAS and ATP5E, the bottom panel displays the most biologically relevant or consensus transcript based on the Ensembl database. The red line on the ideogram, bottom of the figure, represents the location of GNAS and ATP5E on chromosome 20.

Figure 4

Differentially methylated region on chromosome 12 where average (pre- and post-SV) methylation levels differed across the three IA progression phenotypes. Description. Region on chromosome 12 loc 2943902 to 2944481 (μDMR4) where average DNA methylation (DNAm) levels, post- and pre-SV, differed across groups. In the top panel, each dot represents the average DNAm value (y-axis) at each of the CpG sites included μDMR4. The x-axis represents the position (mb) of the CpGs within the region. All DNAm values were adjusted for age, sex, cell proportions, and genetic ancestry. The dashed lines represent the average methylation value within each group across the entire region. The middle panel represents the location of the region (black solid square) relative to the closest genes, ITFG2 and NRIP2 (red solid squares). There are multiple known isoforms for ITFG2 and NRIP2, the figure displays the most biologically relevant or consensus transcript based on the Ensembl database. The red line on the ideogram, bottom of the figure, represents the location of ITFG2 and NRIP2 on chromosome 12.

Figure 5

Differentially changing region on chromosome 6 (post- vs pre-SV) that was positively correlated with changes in lipid metabolites (post- vs pre-SV). Description: Region on chromosome 6 loc 170597377 to 170597899 (ΔDMR8) where the change in DNA methylation (DNAm) post- vs pre-SV differed across groups. In the top left (A), each dot represents the within group slopes (y-axis) or changes in methylation m-values between the post-SV and pre-SV visit at each of the CpG sites included ΔDMR 8. The x-axis represents the position (mb) of the CpGs within the region. Positive values indicate DNAm values increased following IA seroconversion whereas negative values indicate DNAm decreased following IA seroconversion. The dashed lines represent the average slope value within each group across the entire region. The top right (B) represents the association between DMR wide DNAm captured by the 1^st PC (x-axis) and changes in metabolite values (y-axis) between the post- and pre-SV visits. DNAm and metabolite expression values have been standardized to facilitate the interpretation of the slope as a 1 standard deviation increase in the change in metabolite levels between the post- and pre-SV visits per 1 standard deviation increase in the change in methylation between post- and pre-SV visits. The bottom panels (C, D) represent the average metabolite levels and corresponding 95% confidence intervals within the three groups pre- and post-SV. All DNAm and metabolite values were adjusted for age, sex, and cell proportions.