Abstract: SA-OR112

Systems Methylome Analysis Identifies CTCF Regulated Pathways in Disease Progression to Diabetic Nephropathy

Session Information

Category: Diabetes

  • 502 Diabetes Mellitus and Obesity: Clinical

Authors

  • Khurana, Ishant, Central Clinical School, Monash University, Melbourne, Victoria, Australia
  • Cooper, Mark E., Central Clinical School, Monash University, Melbourne, Victoria, Australia
  • Groop, Per-Henrik, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  • El-Osta, Assam, Central Clinical School, Monash University, Melbourne, Victoria, Australia

Group or Team Name

  • Finnish Diabetic Nephropathy study
Background

Despite extensive GWAS by multiple groups and consortia, including the The Finnish Diabetic Nephropathy Study (FinnDiane), only a few genes that explain <5% of susceptibility to nephropathy in type 1 diabetes (T1D) have been identified. With increased awareness of epigenetics as it pertains to human disease, there remains scepticism whether the signalling pathways in diabetes are an association or represent a direct pathogenic state. Therefore, we used a global approach to characterize methylation-mediated function in clinical samples combined with integrative molecular studies to define mechanisms implicated in hyperglycemia and diabetes.

Methods

Leukocyte DNA isolated from case and control groups were fragmented and DNA precipitated using methyl-CpG capture followed by massive parallel sequencing (methyl-seq) . We have mapped the methylome of (FinnDiane) participants selected based on the presence or absence diabetic nephropathy. The case group included 25 participants with T1D characterized into three groups, normoalbuminuria (Normo), macroalbuminuria (Macro) and end stage renal disease (ESRD). The control group consisted of 14 non-diabetic participants with no proteinuria.

Results

We show for the first time DNA methylation sequence changes derived from leukocytes for genes important in insulin signaling, integrin interactions and lipid metabolism. Methylation sequencing identified mechanistic target of rapamycin (mTOR) gene regulation was subject to differential CpG methylation in the genebody at CTCF binding sites. Ex vivo cell experiments confirm transition from normal to high glucose conditions reduced DNA methylation and increased mTOR gene expression. The significance of DNA methylation on mTOR was also validated using the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5adC). We show exon-specific mTOR expression is DNA methylation dependent and hypothesize alternative splicing of mTOR is mediated by Pol II pausing conferred by DNA methylation. CTCF binding is dependent on DNA methylation and regulates mTOR in primary cells stimulated by hyperglycemia and, 5adC.

Conclusion

These results highlight glucose-derived changes to CTCF binding sites are sensitive to loss-of-methylation with gain-of-function of mTOR in diabetes with strengthens the evidence base against DNA methylation changes just being an epiphenomenon.

Funding

  • Government Support - Non-U.S.