DNA Methylation and Autoimmune Thyroid Diseases
Condition: Hashimoto Thyroiditis; Graves Disease
MeSH terms: Graves Disease; Thyroid Diseases; Thyroiditis; Hashimoto Disease
Study type: Observational

Summary: 
      Hashimoto Thyroiditis (HT) and Graves Disease (GD) are known to be caused by abnormal immune
      response against self cells and tissues. Epigenetics is a novel field of biology studying the
      mechanisms by which the environment interacts with the genotype to produce a variety of
      phenotypes through modifications to chromatin that do not directly alter the DNA sequence. A
      very limited number of epigenetic studies have been published in patients with HT and GD so
      far. Therefore, the purpose of this study is to analyze DNA methylation status in White Blood
      Cells (WBCs) within the promoter regions of genomic sites that have been previously
      identified as susceptibility loci or sites for autoimmune thyroid disease, such as the CD40L,
      FOXP3, CTLA4, PTPN22, IL2RA, FCRL3 and HLADRB1 genes.
    

Detailed description: 
      Hashimoto Thyroiditis (HT) and Graves Disease (GD) are known to be caused by abnormal immune
      response against self cells and tissues. HT involves a cell-mediated autoimmune destruction
      of the thyroid leading to hypothyroidism. GD is caused by a process in which immune cells
      make stimulating antibodies against the thyroid stimulating hormone (TSH) receptor on the
      thyroid gland, thus leading to hyperthyroidism. Although there is substantial evidence that
      genetic factors increase the risk for developing autoimmune diseases, monozygotic twins still
      remain discordant for disease (disease concordance is never 100%), thus suggesting a role for
      environmental factors and epigenetics.

      Epigenetics is a novel field of biology studying the mechanisms by which the environment
      interacts with the genotype to produce a variety of phenotypes through modifications to
      chromatin that do not directly alter the DNA sequence. These modifications have been
      associated with altered gene expression and silencing of repetitive elements and can be
      inherited mitotically. Epigenetic mechanisms include DNA methylation, histone modifications,
      or miRNA post-transcriptional regulation. DNA methylation involves the covalent addition of a
      methyl group to the carbon-5 position in the CpG dinucleotide from the methyl donor
      S-adenosylmethionine and is mediated by a group of enzymes called DNA methyltransferases
      (DNMTs). CpG dinucleotides are typically grouped together in regions known as CGIs (islands).
      CGIs can be found in the promoter regions of genes, and CpG methylation of these gene
      promoters is associated with transcriptional silencing. In contrast, hypermethylated genes
      have been found to be transcriptionally active.

      A very limited number of epigenetic studies have been published in patients with HT and GD so
      far. Therefore, the purpose of this study is to analyze DNA methylation status in White Blood
      Cells (WBCs) within the promoter regions of genomic sites that have been previously
      identified as susceptibility loci or sites for autoimmune thyroid disease, such as the CD40L,
      FOXP3, CTLA4, PTPN22, IL2RA, FCRL3 and HLADRB1 genes.

      Initially, recruitment of patients and controls as well as blood sample collection will be
      done. A complete physical examination will also be performed in all participants included in
      the study, and a detailed personal, family, gestational and perinatal history will be
      obtained as well before inclusion. Blood samples by all participants will be collected and
      centrifuged and then White Blood Cells (WBCs), plasma and serum will be separated and stored
      in a deep freezer.

      Laboratory analyses will follow. DNA will be isolated from peripheral leukocytes using the
      QIAamp DNA Blood Mini Kit, according to the manufacturer's instructions. It will then be
      treated with sodium bisulfite using the Zymo EZ DNA Methylation-Gold Kit, again according to
      the manufacturer's protocol. Therefore, unmethylated cytosines will be converted into
      uracyls, whereas methylated cytosines will remain unchanged. Quantification of the
      methylation status of DNA at the gene promoter regions under study will be made, using
      specific primers that detect modified DNA, by real-time PCR and analysis of the melting
      curves of the selected fragments of DNA. Amplicons will also be analyzed by electrophoresis
      and visualized by ultraviolet trans-illumination.

      An electronic Data Base will be constructed and Statistical Analysis will follow. Results and
      Conclusions will be published in peer-review journals and presented in International
      Meetings.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        Children and adolescents of Greek origin, aged 4-18 years old with a diagnosis of Hashimoto
        Thyroiditis and Graves Disease as well as healthy controls.
      
Biospecimen description: 
      Blood samples will be collected and centrifuged and then leukocytes will be separated. DNA
      will then be isolated from peripheral leukocytes using the QIAamp DNA Blood Mini Kit.