Methylation of DNA in Children and Adolescents With Type 1 Diabetes Mellitus (METHYLDIAB)
Condition: Type 1 Diabetes Mellitus
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 1
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        Children and adolescents of Greek origin, aged 2-18 years old with a diagnosis of Type 1
        Diabetes Mellitus as well as healthy controls age and gender matched.
      
Biospecimen description: 
      DNA from white blood cells from peripheral blood samples
    
Keywords: INS gene; methylation; next generation sequencing