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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 CD40, FOXP3, CTLA4, PTPN22, CD25, and TPO genes.
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 CD40, FOXP3, CTLA4, PTPN22, CD25, and TPO 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 standard phenol chloroform technique. It will then be treated with sodium bisulfite using the Zymo EZ DNA Methylation-Gold Kit, according to the manufacturer's protocol. Therefore, unmethylated cytosines will be converted into uracyls, whereas methylated cytosines will remain unchanged. Afterwards, a PCR reaction will be performed, targeting specific sequences in the promoter region of specific gene promoters (using specific primers). Amplicons will then be analyzed by electrophoresis and visualized by ultraviolet trans-illumination, and PCR products will be purified using Millipore Centrifugal Filters for DNA purification. A sequence analysis will also be performed in order to quantitatively describe the methylation status at the genetic loci under study.
Finally, online bioinformatics resources will be employed for aligning the obtained sequences (e.g., BLAST - http://blast.ncbi.nlm.nih.gov/Blast.cgi) and subsequently identifying their methylated and unmethylated CpGs sites. In case more flexibility and automation is needed, bioinformatics scripts will be developed from scratch to accomplish the aforementioned tasks based on powerful, open-source bioinformatics software libraries (e.g., Biopython - http://biopython.org/). 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
Unit of Pediatric Endocrinology, Diabetes and Metabolism-4th Department of Pediatrics, Medical School of Aristotle University of
Active, not recruiting
Aristotle University Of Thessaloniki
Published on BioPortfolio: 2015-07-09T08:38:24-0400
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