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DNA methylation of cytosine-guanine sites (CpGs) is associated with type 1 diabetes (T1D). The sequence of methylated and non-methylated sites in a specific genetic region constitutes its methyl-haplotype. The aim of the present study was to identify insulin gene promoter (IGP) methyl-haplotypes among children and adolescents with T1D and suggest a predictive model for the discrimination of cases and controls according to methyl-haplotypes. A total of 40 individuals (20 T1D) participated. The IGP region from peripheral whole blood DNA of 40 participants (20 T1D) was sequenced using next-generation sequencing, sequences were read using FASTQ files and methylation status was calculated by python-based pipeline for targeted deep bisulfite sequenced amplicons (ampliMethProfiler). Methylation profile at 10 CpG sites proximal to transcription start site of the IGP was recorded and coded as 0 for unmethylation or 1 for methylation. A single read could result in '1111111111' methyl-haplotype (all methylated), '000000000' methyl-haplotype (all unmethylated) or any other combination. Principal component analysis was applied to the generated methyl-haplotypes for dimensionality reduction, and the first three principal components were employed as features with five different classifiers (random forest, decision tree, logistic regression, Naive Bayes, support vector machine). Naive Bayes was the best-performing classifier, with 0.9 accuracy. Predictive models were evaluated using receiver operating characteristics (AUC 0.96). Methyl-haplotypes '1111111111', '1111111011', '1110111111', '1111101111' and '1110101111' were revealed to be the most significantly associated with T1D according to the dimensionality reduction method. Methylation-based biomarkers such as IGP methyl-haplotypes could serve to identify individuals at high risk for T1D. [ABSTRACT FROM AUTHOR]

The insulin (INS) gene is the one of the most important genes involved in the pathogenesis of Type 1 Diabetes (T1D) after the Major Histocompatibility Complex genes. Studies addressing the issue of hyper- or hypo-methylation status of the INS gene promoter have reported inconsistent results. The majority of studies showed hypomethylation; however a few studies have shown hypermethylation at specific cytosine-guanosine (CpG) sites in the promoter region of the INS gene. The aim of the present study was to analyze the methylation status of the promoter region of the INS gene in Greek children and adolescents with T1D. A total of 20 T1D participants (mean diabetes duration of 6.15±4.12 years) and 20 age- and sex-matched controls were enrolled in the present study. DNA was isolated from whole blood samples, modified using sodium bisulfite and analyzed using PCR and electrophoresis. DNA was then pooled with highly reactive supermagnetic beads at similar molar quantities, submitted for library construction and finally sequenced using next-generation sequencing. The methylation profile at 10 CpG sites around the transcription start site (TSS) of the INS promoter was analysed and expressed as the mean ± standard deviation. The overall mean methylation in patients with T1D did not differ compared with the healthy controls. There was a statistically significant difference between the two groups in hypermethylation at position -345 (P=0.02), while a trend (P=0.06) at position -102 was observed. According to the results of the present study, increased methylation in the INS gene promoter at specific CpG sites around the TSS were already present in childhood T1D. These data may possibly serve as a guide towards the identification of a methylation pattern for detection of development of T1D in genetically predisposed children. [ABSTRACT FROM AUTHOR]