DNA Methylation and Autoimmune Thyroid Diseases

Completed

• Conditions: Graves Disease; Hashimoto Thyroiditis

• Registration Number: NCT02491567
• Lead Sponsor: Aristotle University Of Thessaloniki

Brief 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.

Study Timeline

• Study Start: 2014-09
• Study First Submitted: 2014-12-23
• Study First Submitted QC: 2015-07-07
• Study First Posted: 2015-07-08
• Primary Completion: 2016-09
• Study Completion: 2018-04
• Status Verified: 2019-09
• Last Update Submitted: 2019-09-24
• Last Update Posted: 2019-09-26

Recruitment & Eligibility

• Status: COMPLETED
• Sex: All
• Target Recruitment: 110

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

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Primary Outcome Measures

Name	Time	Method
DNA methylation status of CpGs within gene promoters	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).

Secondary Outcome Measures

Name	Time	Method
Sex	1 day	Male or female
Body mass index	1 day	Weight in kg / height in m \* height in m
Treatment dose	1 day	Dose of Levothyroxine/thiamazole per kg of body weight /per day (if applicable)
Previous births	1 day	Number of previous births
Delivery type	1 day	Cesarean section or vaginal delivery
Thyroid volume	1 day	Volume of the thyroid gland in total (both lobes)
Folic acid	1 day	Levels of folic acid in blood
History of medications	1 day	Number of previous medications per year
Month of birth	1 day	Month of birth (from January to December)
Gestation duration	1 day	Duration of pregnancy
Maternal alcohol consumption during pregnancy	1 day	Total number of glasses of alcohol consumption per day during pregnancy (if applicable)
History of phototherapy	1 day	History of phototherapy during neonatal period (or not)
Age	1 day	Age of each participant
History of infections	1 day	Number of previous febrile viral /bacterial infections per year
Age of disease onset	1 day	Age of disease diagnosis
Antibodies titre	1 day	Titre of antiTPO, antiTg, anti-TSI antibodies in blood
IgA, IgG, IgM, IgE immunoglobulins	1 day	Levels of IgA, IgG, IgM, IgE immunoglobulins in blood
Other autoimmune diseases	1 day	Diagnosis of co-existing autoimmune disease (except autoimmune thyroid disease)
Maternal febrile infection (during pregnancy)	1 day	Diagnosis of maternal febrile infection (during pregnancy) or not
APGAR score	1 day	APGAR score at 1st and 5th min of life
Pubertal stage	1 day	Prepubertal or pubertal stage
B12	1 day	Levels of B12 in blood
Family history of autoimmune thyroid (or other) disease	1 day	Family history of autoimmune thyroid (or other) disease or not
Parental educational level	1 day	Elementary school, high school or university graduate
Parental smoking	1 day	Total number of cigarettes per day during their child's life separately for each parent (if applicable)
Medications during pregnancy	1 day	Number of medications of any type received during pregnancy (if applicable)
Maternal smoking during pregnancy	1 day	Total number of cigarettes per day during pregnancy (if applicable)
Pre-eclampsia (during pregnancy)	1 day	Diagnosis of pre-eclampsia (during pregnancy) or not
Type of Residence	1 day	Urban or rural residence
Birth weight	1 day	Birth weight
Gestational diabetes (during pregnancy)	1 day	Diagnosis of gestational diabetes (during pregnancy) or not
Vaginal bleeding (during pregnancy)	1 day	Presence of vaginal bleeding (during pregnancy) or not
Duration of breastfeeding	1 day	Duration of breastfeeding until discontinuance

Trial Locations

Locations (1)

Unit of Pediatric Endocrinology, Diabetes and Metabolism-4th Department of Pediatrics, Medical School of Aristotle University of Thessaloniki; 🇬🇷 Thessaloniki, Greece