Genome-wide DNA methylation analysis for diabetic nephropathy in type 1 diabetes mellitus

Abstract

Background: Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease.

Methods: We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes.

Results: Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy.

Conclusion: This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy.

Figure 1

Average β values. Figure 1a (top): Controls, Figure 1b (bottom): Cases - Average β values across array.

Figure 2

Fraction of variation associated with principal components. Fraction of variation associated with principal components after adjustment for the top component which captures the inherent bi-modality of the methylation distribution. Red denotes the observed values, black denotes those obtained by randomly scrambling up the data matrix. Each black dot represents the value of 10 distinct randomisations, which all yield the same value as singular values represent measures of global variation which themselves are invariant under a global randomisation of the data.

Figure 3

Correlation of principle components to phenotype/experimental factors. Correlation of principal components (top component removed as it did not correlate with any phenotype) to phenotypes of interest and experimental factors: BS conversion (bisulphite conversion, C1(green), C2(blue)), Sex, Cohort, Chip, Batch, Age at Draw (Age when sample taken), Duration T1D, Age at Diagnosis, CC (time to onset of NP) Colour key: dark red = p value < 10^-10, red = p value < 10^-5, orange = p value < 10^-2, pink = p value < 0.05, white = p value > 0.05.

Figure 4

Maximum and minimum β values for Chromosome 1. Maximum and minimum β values across the 2903 probed CpGs located on Chromosome 1 for Controls and Cases.

Figure 5

Nephropathy and duration type 1 diabetes p values. Histogram of p-values after explicit correction for all potentially confounding factors reveals an excess of significant p values for CpGs correlated with Nephropathy and duration of Type 1 Diabetes.

Figure 6

Z score values for cg07341907. Box plot for adjusted comparison of methylation Z-score values for the UNC13B promoter CpG:cg07341907 on controls (0) and cases (1).