Genome-wide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood

Abstract

Inter-individual DNA methylation variations were frequently hypothesized to alter individual susceptibility to Type 2 Diabetes Mellitus (T2DM). Sequence-influenced methylations were described in T2DM-associated genomic regions, but evidence for direct, sequence-independent association with disease risk is missing. Here, we explore disease-contributing DNA methylation through a stepwise study design: first, a pool-based, genome-scale screen among 1169 case and control individuals revealed an excess of differentially methylated sites in genomic regions that were previously associated with T2DM through genetic studies. Next, in-depth analyses were performed at selected top-ranking regions. A CpG site in the first intron of the FTO gene showed small (3.35%) but significant (P = 0.000021) hypomethylation of cases relative to controls. The effect was independent of the sequence polymorphism in the region and persists among individuals carrying the sequence-risk alleles. The odds of belonging to the T2DM group increased by 6.1% for every 1% decrease in methylation (OR = 1.061, 95% CI: 1.032-1.090), the odds ratio for decrease of 1 standard deviation of methylation (adjusted to gender) was 1.5856 (95% CI: 1.2824-1.9606) and the sensitivity (area under the curve = 0.638, 95% CI: 0.586-0.690; males = 0.675, females = 0.609) was better than that of the strongest known sequence variant. Furthermore, a prospective study in an independent population cohort revealed significant hypomethylation of young individuals that later progressed to T2DM, relative to the individuals who stayed healthy. Further genomic analysis revealed co-localization with gene enhancers and with binding sites for methylation-sensitive transcriptional regulators. The data showed that low methylation level at the analyzed sites is an early marker of T2DM and suggests a novel mechanism by which early-onset, inter-individual methylation variation at isolated non-promoter genomic sites predisposes to T2DM.

Figure 1.

A flowchart of the multistep study design. Pool-based assessments of CpG sites in methyl-sensitive restriction enzyme (MSRE) sites within microarray-probed DNA fragments (i), allowed genome-scale evaluation of differentially methylated regions (DMRs) (ii). Following these steps, deep bisulfite sequencing was utilized to verify particular case–control-differentiating sites (iii). Finally, effect size, sensitivity and mechanistic aspects were explored through individual-level analyses (iv).

Figure 2.

Case–control differentially methylated regions (DMRs) in T2DM-associated genomic blocks. The chromosomal locations and sizes of the 30 established T2DM-associated genomic linkage disequilibrium (LD) blocks, and their coverage by the microarray-based assay are shown. The indicated genes provide the common reference in the literature, and not the complete list of genes residing in these regions. Unshaded boxes indicate the coverage of these regions by microarray-probed 10 kb ‘informative’ windows (50% overlapped). Gray boxes indicate DMRs, and black boxes indicate stringent DMRs. Asterisks indicate LD blocks that were significantly (P < 0.05) enriched by stringent DMRs following correction to the number of informative windows within the block. Note that the indicated genes are only reference to the block identity as given in the study of Voight et al. (2), not necessarily the actual T2DM gene affected by the disease-associated (genetic or epigenetic) polymorphisms in the region.

Figure 3.

Sequence-independent differential methylation. (A) Map of a DNA fragment from the first intron of the FTO gene, containing a CpG site and a T2DM/obesity-associated polymorphic site. (B–E) The results of ultra-deep sequencing of this fragment among two case and two control pools, allowing co-assessment of genotype and epigenotype differences. (B) Frequencies of the A allele among case and controls pools. (C) Methylation levels of sequenced DNA molecules carrying the A or the G alleles. The P-value of the difference between the case and control pools is indicated. (D) Methylation levels of case and control pools (both alleles). (E) Methylation levels of molecules from the case or the control pools carrying the A allele. The data show that despite the higher frequency of the A risk allele among the cases (B), and the higher methylation level associated with the risk allele (C), cases are more methylated than controls (D), and this trend persists among DNA molecules carrying the A allele (E). Thus, the hypomethylation of the patients is sequence-independent.

Figure 4.

Individual-level analyses of the FTO methylation site. Methylation levels of the methylation site presented in Figure 3 were evaluated in patients and controls by quantitative pyrosequencing. (A) Frequencies and Gaussian expectations of methylation levels among 198 T2DM patients and 233 controls. The P-value of the methylation difference between cases and control following adjustment for the effect of gender is indicated. Insert: Receiver-operating characteristic (ROC) analysis of the same individuals. The area under the curve (AUC) is a measurement of the sensitivity, as expressed by the ratio of true-positive to false-positive rates. The dashed line represents the null hypothesis of no prediction. (B) ROC analyses as above, but for males and females separately. (C) Methylation levels of case and control individuals categorized by gender. (D) Methylation levels of case and control individuals categorized by age.

Figure 5.

Case–control differentiation is not due to body mass index BMI. (A) BMI of control individuals as a function of their methylation level. (B) Methylation levels of case and control individuals categorized by BMI. BMI data were available for 365 out of the 431 individuals presented in Figure 4.

Figure 6.

Abnormal methylation levels precede the onset of disease manifestations. (A) Scheme of the Jerusalem LRC longitudinal (cohort) study: Out of 515 individuals with normal glucose metabolism at age 30, 62 had, by age 43, developed IGM, defined as fasting plasma glucose levels ≥110 mg/dl and/or ≥140 mg/dl 2h after a 75 g oral glucose load, including eight that fulfilled clinical criteria for T2DM; 114 developed impaired fasting glucose (IFG, fasting glucose 100–109 mg/dl); and 339 remained normal (fasting glucose <100 mg/dl and post-challenge <140 mg/dl). (B) Methylation levels at baseline (age 30) of 58 incidence cases of IGM (including seven with diabetes) and 64 randomly selected normal controls. P-value (logistic regression, one-sided) was obtained following adjustment for gender and lymphocyte count.

Figure 7.

Differentially methylated regions (DMRs) are co-localized with transcription enhancers and binding sites for methylation-sensitive transcription factors. (A) A map of the FTO LD block showing (from top to bottom) USF1/2 binding sites, probed (gray boxes) and differentially methylated (black boxes) DNA fragments, DMRs (black line), H3K4Me1 chromatin marks (deposited data in Mikkelesen et al. (#43)), T2DM-associated polymorphic sequences and a genetic linkage map. CpG sites that showed significant differences between case and control pools (hypomethylation in patients) are also indicated. (B) Same as in panel (A), but for the TCF7L2 block. (C) Average levels of H3K4Me1 enhancer marks in 10 kb windows along the T2DM LD blocks, in windows without or with USF1/2 binding sites, and in DMRs. To allow evaluation on a common scale, the H3K4Me1 levels of six cell lines were normalized to a common mean and variance. (D) The ratio of observed/expected numbers of binding sites (expectation is based on the frequency across the genome) in all genomic DMRs, in LD block windows, in LD block windows enriched with H3K4Me1 marks and in LD block DMRs. The data show that DMRs within the LD blocks are significantly enriched with binding sites for methylation-sensitive transcription factors. The presented data refer to all DMRs and similar enrichments were observed for the stringent DMRs (Supplementary Material, Fig. S5). USF1/2 and MYCN shared the E-box sequence-binding motif (CACGTG), while E2F binds a different core motif (^C/_GGCGC). USF1/2 were the only methylation-sensitive transcription factors that showed enrichment P-values <0.001 in both DMRs and stringent DMR analyses. Asterisks denote P < 0.05; double asterisks denote P < 0.01.