ESPE Abstracts (2014) 82 LBP-D--3-1014

CpG Methylation Changes within the INS, HLA-G and PTPN-22 Promoters in Childhood Type 1 Diabetes

Ioannis Kyrgiosa, Konstantina Mouzakia, Theodoros Spandonidisb, Aikaterini Fragoub, Georgios Tzimagiorgisb, Maria Eboriadou-Petikopouloua & Assimina Galli-Tsinopouloua

a4th Department of Pediatrics, Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece; bLaboratory of Biological Chemistry, Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

Background: Alterations in DNA methylation status of specific genetic loci may affect gene expression, thus leading to autoimmunopathies.

Objective and hypotheses: This study aimed to investigate possible differences in DNA methylation pattern between type 1 diabetes mellitus (T1DM) youngsters and healthy controls.

Method: Ten T1DM participants and 10 age-/gender-matched controls were enrolled. DNA was extracted from white blood cells using the standard phenol chloroform technique. Genomic DNA (800 ng) was modified using the EZ DNA Methylation-Gold Kit. Treatment with sodium bisulfite converts unmethylated cytosines into uracyls, whereas methylated cytosines remain unchanged. PCR reaction was performed in a total volume of 50 μl targeting a specific sequence of the 3 genes promoters HLA-G 441, INS 415, PTPN-22 418 bp sequence, respectively. The primers used to amplify the promoters sequences were: HLA-G forward 5′-GGGAGGTAGGGAGTTTAGTTTA3′, reverse 5′-CCATAACCACCATCCTTAAC3′, INS forward 5′-TTTGGGGATAGGGGTTTGGGGATAGTA3′, reverse 5′-CCTCTTCTAATACAACCTATCCTAAAAAACTAAAAACTAC3′ and PTPN-22 forward 5′-TGATGGAATGGAATTTTAGTT-AAG3′, reverse 5′-CACCAAAAATTCATTAACAAACTCC3′. Amplicons were analyzed by electrophoresis (1% agarose gel stained with ethidium bromide) and visualized by ultraviolet trans-illumination. PCR products were purified using Millipore Centrifugal Filters for DNA Purification and sequenced to identify any differences in DNA methylation.

Results: Methylation profile of 16, 4 and 9 CpGs of the HLA-G, PTPN-22 and INS promoter respectively, was analyzed. No difference between T1DM cases and controls concerning the CpGs of all studied promoters was found, although a trend towards increased levels of methylation was observed in a number of CpGs of the INS promoter.

Conclusion: These preliminary data suggest that a tendency for increased methylation in INS promoter already exists in T1DM in childhood. Studies with greater number of participants are needed to confirm these findings.

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