Background: Prolonged hyperglycemia causes micro- and macrovascular complications, which are the main cause of diabetes-related morbidity and mortality. Growing evidence suggests that prolonged hyperglycemia influences the development of vascular complications through epigenetic mechanisms, such as DNA methylation, histone modifications, and microRNA. DNA methylation was already associated with diabetes-related complications of type 1 diabetes (T1D), such as diabetic retinopathy, diabetic nephropathy, and diabetic foot. DNA methylation was not yet studied in individuals with T1D without clinical manifestation of diabetes-related complications with different levels of glycemic control.
Methods: 146 participants with type T1D for at least 5 years, aged between 10 and 21, and without clinical manifestation of diabetes-related complications were included. Their glycemic control was assessed with the mean values of glycated hemoglobin (HbA1c) and glycemic variability (GV), which was determined as coefficient of variation. Based on their values, the participants were divided into groups that were then compared one to another (HbA1c < 7% vs. HbA1c > 8% and GV < 40% vs. GV > 50%). The participants DNA was isolated from the whole blood and pooled together according to their mean values of HbA1c and GV. Differentially methylated regions were determined with MEDIP on pooled samples. For the conformation of identified regions, Cas9 enrichment and third-generation of sequencing was performed on pooled samples of participants with the most extreme values of HbA1c and GV. Finally, third-generation of sequencing was used to determine differentially methylated sites and regions on whole genome on a pooled sample of participants with most extreme values of HbA1c.
Results: With MEDIP, 14 differentially methylated regions were detected between groups HbA1c<7% and HbA1c>8%, including 11 hypomethylated and 3 hypermethylated according to group HbA1c>8%. Between groups GV<40% and GV>50%, 4 differentially methylated regions were identified, two hypomethylated and two hypermethylated according to CV>50%. These regions were not conformed on pooled samples of participants with the most extreme values of HbA1c and GV. With whole genome third-generation sequencing, 8385 differentially methylated CpG sites were identified between groups with the most extreme values of HbA1c. These sites were annotated to 1802 genes. Several of these genes have previously already been associated with diabetes-related complications. Genes annotated to hypomethylated sites were enriched in 48 signaling pathways. Merging neighboring CpG sites resulted in 80 differently methylated regions.
Conclusions: Prolonged hyperglycemia in individuals is associated with differentially methylated sites and regions located in critical genes and pathways with a known role in diabetes-related complications. Further research could reveal the exact CpG sites that could be used as reliable biomarkers for detecting individuals with higher risk for development of diabetes-related complications before the manifestation of clinical symptoms, even in individuals with T1D and strictly managed glycemic control.
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