DNA Methylation and Autoimmune Thyroid Diseases (THYRODNA)
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|ClinicalTrials.gov Identifier: NCT02491567|
Recruitment Status : Completed
First Posted : July 8, 2015
Last Update Posted : September 26, 2019
|Condition or disease|
|Hashimoto Thyroiditis Graves Disease|
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.
|Study Type :||Observational|
|Actual Enrollment :||110 participants|
|Official Title:||Study of DNA Methylation in Children and Adolescents With Autoimmune Thyroid Diseases|
|Actual Study Start Date :||September 2014|
|Actual Primary Completion Date :||September 2016|
|Actual Study Completion Date :||April 2018|
Hashimoto Thyroiditis (HT)
Children and adolescents with Hashimoto thyroiditis either hypothyroidic or euthyroidic.
Graves Disease (GD)
Children and adolescents with Graves Disease both those on remission and under antihyroid medication.
Healthy individuals matched for gender and age without 1) any autoimmune disease 2) family history of autoimmune disease in the first degree relatives
- DNA methylation status of CpGs within gene promoters [ Time Frame: 1 month ]Percentage of DNA methylation of CpGs within the CD40L, FOXP3, CTLA4, PTPN22, IL2RA, FCRL3 and HLADRB1 promoter genes in White Blood Cells (WBCs).
- Age [ Time Frame: 1 day ]Age of each participant
- Age of disease onset [ Time Frame: 1 day ]Age of disease diagnosis
- Sex [ Time Frame: 1 day ]Male or female
- Body mass index [ Time Frame: 1 day ]Weight in kg / height in m * height in m
- Pubertal stage [ Time Frame: 1 day ]Prepubertal or pubertal stage
- Antibodies titre [ Time Frame: 1 day ]Titre of antiTPO, antiTg, anti-TSI antibodies in blood
- Thyroid volume [ Time Frame: 1 day ]Volume of the thyroid gland in total (both lobes)
- Treatment dose [ Time Frame: 1 day ]Dose of Levothyroxine/thiamazole per kg of body weight /per day (if applicable)
- B12 [ Time Frame: 1 day ]Levels of B12 in blood
- Folic acid [ Time Frame: 1 day ]Levels of folic acid in blood
- IgA, IgG, IgM, IgE immunoglobulins [ Time Frame: 1 day ]Levels of IgA, IgG, IgM, IgE immunoglobulins in blood
- History of infections [ Time Frame: 1 day ]Number of previous febrile viral /bacterial infections per year
- History of medications [ Time Frame: 1 day ]Number of previous medications per year
- Other autoimmune diseases [ Time Frame: 1 day ]Diagnosis of co-existing autoimmune disease (except autoimmune thyroid disease)
- Family history of autoimmune thyroid (or other) disease [ Time Frame: 1 day ]Family history of autoimmune thyroid (or other) disease or not
- Parental educational level [ Time Frame: 1 day ]Elementary school, high school or university graduate
- Type of Residence [ Time Frame: 1 day ]Urban or rural residence
- Parental smoking [ Time Frame: 1 day ]Total number of cigarettes per day during their child's life separately for each parent (if applicable)
- Previous births [ Time Frame: 1 day ]Number of previous births
- Month of birth [ Time Frame: 1 day ]Month of birth (from January to December)
- Delivery type [ Time Frame: 1 day ]Cesarean section or vaginal delivery
- Birth weight [ Time Frame: 1 day ]Birth weight
- Gestation duration [ Time Frame: 1 day ]Duration of pregnancy
- Medications during pregnancy [ Time Frame: 1 day ]Number of medications of any type received during pregnancy (if applicable)
- Maternal smoking during pregnancy [ Time Frame: 1 day ]Total number of cigarettes per day during pregnancy (if applicable)
- Maternal alcohol consumption during pregnancy [ Time Frame: 1 day ]Total number of glasses of alcohol consumption per day during pregnancy (if applicable)
- Pre-eclampsia (during pregnancy) [ Time Frame: 1 day ]Diagnosis of pre-eclampsia (during pregnancy) or not
- Gestational diabetes (during pregnancy) [ Time Frame: 1 day ]Diagnosis of gestational diabetes (during pregnancy) or not
- Vaginal bleeding (during pregnancy) [ Time Frame: 1 day ]Presence of vaginal bleeding (during pregnancy) or not
- Maternal febrile infection (during pregnancy) [ Time Frame: 1 day ]Diagnosis of maternal febrile infection (during pregnancy) or not
- Duration of breastfeeding [ Time Frame: 1 day ]Duration of breastfeeding until discontinuance
- History of phototherapy [ Time Frame: 1 day ]History of phototherapy during neonatal period (or not)
- APGAR score [ Time Frame: 1 day ]APGAR score at 1st and 5th min of life
Biospecimen Retention: Samples With DNA
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT02491567
|Unit of Pediatric Endocrinology, Diabetes and Metabolism-4th Department of Pediatrics, Medical School of Aristotle University of Thessaloniki|
|Thessaloniki, Greece, 56403|
|Principal Investigator:||Assimina Galli-Tsinopoulou, Professor||Medical School, Aristotle University of Thessaloniki|