Methylation of DNA in Children and Adolescents With Type 1 Diabetes Mellitus (METHYLDIAB)

Type 1 Diabetes Mellitus (T1DM) is a well-studied autoimmune disease resulting in insulin deficiency due to selective β-cell destruction. Epigenetics is a novel field of biology studying the inherited changes in deoxyribonucleic acid (DNA) expression which cannot be attributed to base sequence alteration. A relatively limited number of studies are published until now concerning T1DM in children and adolescents addressing epigenetics changes in DNA expression. The purpose of the present study is to analyze the methylation status of DNA within the promoter region of specific susceptibility genes such as Protein tyrosine phosphatase, non-receptor type 22 (PTPN-22), Insulin (INS) and Human leukocyte antigen G (HLA-G) genes.

Type 1 Diabetes Mellitus (T1DM) is a well-studied autoimmune disease resulting in insulin deficiency due to selective β-cell loss. Environmental and genetics factors seem to have a complex interplay in genetically susceptible individuals leading to T1DM development.

Epigenetics is a new field of biology studying the inherited changes in deoxyribonucleic acid (DNA) expression which cannot be attributed to DNA sequence alteration via DNA methylation, histone modification, and micro-RNAs acting as post-transcriptional regulators.

The methylation of cytosine - guanosine dinucleotides (CpGs), located at the promoter region of the genes, play a vital role in transcription and gene expression and is catalyzed at the 5' cytosine position via enzymes called DNA methyltransferases (DNMTs).

A limited number of studies regarding epigenetics in T1DM paediatric patients have been published so far. The purpose of the present study is to investigate the CpG islet methylation pattern in the promoter regions of specific susceptibility genes such as Protein tyrosine phosphatase, non-receptor type 22 (PTPN-22), Insulin (INS) and Human leukocyte antigen G (HLA-G) genes, extracted from White Blood Cells (WBCs) from T1DM and healthy controls children and adolescents .

Twenty patients and twenty age and gender matched controls were recruited. A detailed personal, family, gestational/perinatal history was obtained and a thorough physical examination was performed in all study participants. Both groups and their first grade relatives had no history of other autoimmune diseases.

Parents provided written informed consent for the participation of their children, according to the declaration of Helsinki for research. involving human subjects .

Protocols were approved by Bioethics Committee of School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece (Protocol No 185/30.12.2015).

Whole blood samples were collected from both groups after a 12-hour period of fast and stored immediately at -80 oC.

DNA was extracted from blood samples using the DNA extraction kit QIAamp® DNA Blood Mini Kit (QIAGEN Inc, CA, USA), as suggested by the manufacturer. Isolated samples were quantified spectrophotometrically using the odds ratio (OD ratio) 260/280 (1 OD = 50 μg/ml), (BioPhotometer 6131 Eppendorf AG, Germany). Bisulfite-treatment was conducted on 300ng DNA of each sample by using the EZ DNA Methylation-Gold kit (Zymo Research, Methylation-Gold, USA), as recommended by the manufacturer. Treatment with sodium bisulphate converts unmethylated cytosines into uracils, whereas methylated cytosines remain unchanged under the same stable conditions.

For INS gene promoter amplification, gene-specific primers were used as follows: INS-Forward 5'-TATTTTGGAATTTTGAGTTTATT-3'and INS-Reverse 5'-AACAAAAATCTAAAAACAACAA-3', for PTPN-22-Forward 5'-TTTTGGTTTATGTTGTAGAGT -3΄ and PTPN-22 Reverse 5΄- ATTTTATTTTATTATTTATATGTAA-3' and for HLA-G- Forward 5'-TAGGGAGTTTAGTTTAGGGAT -3' and Reverse 5'- TTAAGGATGGTGGTTATGG -3'.

Additional, overhang adapter sequence was added to the locus-specific primers for the regions to be targeted (Nextera Transposase Adaptors, Illumina), in order of prompt construction of Next Generation Sequencing (NGS) libraries. Polymerase Chain Reaction (PCR) products were amplified on a low temperature ramping instrument, the 9700 thermal cycler (Eppendorf AG No5341, 9600 emulation mode) using the AmpliTaq Gold DNA Polymerase.

After purification of PCR products with highly reactive super magnetic beads NucleoMag NGS Clean-up and Size Select (Macherey-Nagel. Cat. Number 744970.5.), they were pooled at similar molar quantities and submitted for library construction according to manufacturer instructions, Nextera XT DNA Library Preparation kit, Research Illumina.

For NGS paired-end reads were selected at 2 x 250 base pair read length formation, on a platform Illumina's MiSeq.

Sequence analysis reads were carried out using FASTQ files and methylation status was estimated with the tool ampliMethProfiler, a python-based pipeline for targeted deep bisulfite sequenced amplicons.The methylation status was analyzed at ten CpG sites of INS gene promoter, four CpG sites of PTPN-22 gene and nineteen CpG sites of HLA-G gene around the transcriptional start site (TSS).

The sequences and the identification of the methylated and unmethylated sites will be interpreted by bioinformatics scientists.

A Data-Base will be created and all the data will undergo statistical analysis.The results and the conclusions of the present study will be published in peer- review journals and presented in National and International Meetings.

Eligibility Criteria:
Inclusion Criteria for both groups are:

* no history of T1DM or other autoimmune disease of their first degree relatives
* Greek origin (at least for 3 generations back to be Greek)
* sign written consent form

Inclusion Criteria for patients are:

* males and females 2-18 years of age, with the diagnosis of T1DM
* T1DM diagnosis according to the criteria of International Society for Pediatric and Adolescent Diabetes (ISPAD) and ADA (American Diabetes Association)

Inclusion Criteria for Controls are:

* healthy males and females 2-18 years of age, with no consaguinity with the patients