Overview

This trial is active, not recruiting.

Conditions hashimoto thyroiditis, graves disease
Sponsor Aristotle University Of Thessaloniki
Start date September 2014
End date September 2016
Trial size 100 participants
Trial identifier NCT02491567, 62/17-2-2014

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 CD40, FOXP3, CTLA4, PTPN22, CD25, and TPO genes.

United States No locations recruiting
Other Countries No locations recruiting

Study Design

Observational model case control
Time perspective cross-sectional
Arm
Children and adolescents with Hashimoto thyroiditis either hypothyroidic or euthyroidic.
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

Primary Outcomes

Measure
DNA methylation status of CpGs within the promoters of CD40, FOXP3, CTLA4, PTPN22, CD25, and TPO promoter genes in White Blood Cells (WBCs).
time frame: 1 month

Secondary Outcomes

Measure
Age of disease onset
time frame: 1 day
Treatment dose
time frame: 1 day
Antibodies titre
time frame: 1 day
Thyroid volume
time frame: 1 day
Frequency of previous viral /bacterial infections per year
time frame: 1 day
Frequency of previous medications per year
time frame: 1 day
Consumption of additional iodized salt per week
time frame: 1 day
Consumption of alcohol per week
time frame: 1 day
Consumption of caffeine per week
time frame: 1 day
Frequency of smoking per week
time frame: 1 day
Previous exposure to pesticides per year
time frame: 1 day
Previous exposure to adhesives, plastic materials or other chemicals per year
time frame: 1 day
Parental educational level
time frame: 1 day
Urban / rural residence
time frame: 1 day
Frequency of parental smoking per week
time frame: 1 day
Gestation duration
time frame: 1 day
Medications during pregnancy
time frame: 1 day
Frequency of maternal smoking during pregnancy per week
time frame: 1 day
Presence of preeclampsia
time frame: 1 day
Presence of gestational diabetes
time frame: 1 day
Delivery type
time frame: 1 day
Birth weight
time frame: 1 day
APGAR score at 1st and 5th min
time frame: 1 day
Duration of breastfeeding
time frame: 1 day

Eligibility Criteria

Male or female participants from 4 years up to 18 years old.

Inclusion Criteria: For HT: A positive titers of antithyroid peroxidase (anti-TPO) or antithyroglobulin (anti-Tg) antibodies and at least one of: - Abnormal thyroid function that requires substitution treatment with L-thyroxine (TSH > 5 μIU/ml and decreased or normal levels of fT4 or fT3) - Increased volume of thyroid gland (goiter) - Morphological changes on ultrasound of the thyroid gland For GD: - A positive titers of thyroid stimulating antibodies (anti-TSI) and - Decreased TSH levels and increased levels of fT4 or fT3 For Controls: - Otherwise healthy children and adolescents, age- and gender-matched with patients - Absence of previously known chronic disease of autoimmune aetiology or atopy (including those with a history of chronic treatment with antihistamines, anti-inflammatory, corticosteroids or anti-epileptic drugs) - Absence of a family history of autoimmune disease in first-degree relatives Exclusion Criteria: - Not Caucasian origin or affinity among participants - Age of diagnosis above 18 years - Disease duration below 3 months

Additional Information

Official title Study of DNA Methylation in Children and Adolescents With Autoimmune Thyroid Diseases
Principal investigator Assimina Galli-Tsinopoulou, Assoc Prof
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 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.
Trial information was received from ClinicalTrials.gov and was last updated in December 2015.
Information provided to ClinicalTrials.gov by Aristotle University Of Thessaloniki.