DNA Methylation and Autoimmune Thyroid Diseases (THYRODNA)

Study of DNA Methylation in Children and Adolescents With Autoimmune Thyroid Diseases

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.

Study Overview

• Status: Completed
• Conditions: Graves Disease; Hashimoto Thyroiditis

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 Type: Observational
• Enrollment (Actual): 110

Contacts and Locations

Study Locations

• Greece
  • Thessaloniki, Greece, 56403
    • Unit of Pediatric Endocrinology, Diabetes and Metabolism-4th Department of Pediatrics, Medical School of Aristotle University of Thessaloniki

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 2 years to 16 years (Child, Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• 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.

Description

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 Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Cross-Sectional

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort
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.
Controls (C); Healthy individuals matched for gender and age without 1) any autoimmune disease 2) family history of autoimmune disease in the first degree relatives

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
DNA methylation status of CpGs within gene promoters	Percentage of DNA methylation of CpGs within the CD40L, FOXP3, CTLA4, PTPN22, IL2RA, FCRL3 and HLADRB1 promoter genes in White Blood Cells (WBCs).	1 month

Secondary Outcome Measures

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

Collaborators and Investigators

• Sponsor: Aristotle University Of Thessaloniki
• Investigators: Principal Investigator: Assimina Galli-Tsinopoulou, Professor, Medical School, Aristotle University of Thessaloniki

Publications and helpful links

General Publications

• Akirav EM, Lebastchi J, Galvan EM, Henegariu O, Akirav M, Ablamunits V, Lizardi PM, Herold KC. Detection of beta cell death in diabetes using differentially methylated circulating DNA. Proc Natl Acad Sci U S A. 2011 Nov 22;108(47):19018-23. doi: 10.1073/pnas.1111008108. Epub 2011 Nov 9.
• Barrett JC, Clayton DG, Concannon P, Akolkar B, Cooper JD, Erlich HA, Julier C, Morahan G, Nerup J, Nierras C, Plagnol V, Pociot F, Schuilenburg H, Smyth DJ, Stevens H, Todd JA, Walker NM, Rich SS; Type 1 Diabetes Genetics Consortium. Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nat Genet. 2009 Jun;41(6):703-7. doi: 10.1038/ng.381. Epub 2009 May 10.
• Cooper JD, Simmonds MJ, Walker NM, Burren O, Brand OJ, Guo H, Wallace C, Stevens H, Coleman G; Wellcome Trust Case Control Consortium, Franklyn JA, Todd JA, Gough SC. Seven newly identified loci for autoimmune thyroid disease. Hum Mol Genet. 2012 Dec 1;21(23):5202-8. doi: 10.1093/hmg/dds357. Epub 2012 Aug 24.
• Dang MN, Buzzetti R, Pozzilli P. Epigenetics in autoimmune diseases with focus on type 1 diabetes. Diabetes Metab Res Rev. 2013 Jan;29(1):8-18. doi: 10.1002/dmrr.2375.
• Davies TF, Latif R, Yin X. New genetic insights from autoimmune thyroid disease. J Thyroid Res. 2012;2012:623852. doi: 10.1155/2012/623852. Epub 2012 Feb 28.
• Fradin D, Le Fur S, Mille C, Naoui N, Groves C, Zelenika D, McCarthy MI, Lathrop M, Bougneres P. Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes. PLoS One. 2012;7(5):e36278. doi: 10.1371/journal.pone.0036278. Epub 2012 May 2.
• Huber A, Menconi F, Corathers S, Jacobson EM, Tomer Y. Joint genetic susceptibility to type 1 diabetes and autoimmune thyroiditis: from epidemiology to mechanisms. Endocr Rev. 2008 Oct;29(6):697-725. doi: 10.1210/er.2008-0015. Epub 2008 Sep 5.
• Lu Q. The critical importance of epigenetics in autoimmunity. J Autoimmun. 2013 Mar;41:1-5. doi: 10.1016/j.jaut.2013.01.010. Epub 2013 Feb 1.
• MacFarlane AJ, Strom A, Scott FW. Epigenetics: deciphering how environmental factors may modify autoimmune type 1 diabetes. Mamm Genome. 2009 Sep-Oct;20(9-10):624-32. doi: 10.1007/s00335-009-9213-6. Epub 2009 Aug 22.
• Quintero-Ronderos P, Montoya-Ortiz G. Epigenetics and autoimmune diseases. Autoimmune Dis. 2012;2012:593720. doi: 10.1155/2012/593720. Epub 2012 Mar 22.
• Rakyan VK, Beyan H, Down TA, Hawa MI, Maslau S, Aden D, Daunay A, Busato F, Mein CA, Manfras B, Dias KR, Bell CG, Tost J, Boehm BO, Beck S, Leslie RD. Identification of type 1 diabetes-associated DNA methylation variable positions that precede disease diagnosis. PLoS Genet. 2011 Sep;7(9):e1002300. doi: 10.1371/journal.pgen.1002300. Epub 2011 Sep 29.
• Weetman AP. Determinants of autoimmune thyroid disease. Nat Immunol. 2001 Sep;2(9):769-70. doi: 10.1038/ni0901-769. No abstract available.
• Yin X, Latif R, Tomer Y, Davies TF. Thyroid epigenetics: X chromosome inactivation in patients with autoimmune thyroid disease. Ann N Y Acad Sci. 2007 Sep;1110:193-200. doi: 10.1196/annals.1423.021.

Study record dates

Study Major Dates

• Study Start (Actual): September 1, 2014
• Primary Completion (Actual): September 1, 2016
• Study Completion (Actual): April 1, 2018

Study Registration Dates

• First Submitted: December 23, 2014
• First Submitted That Met QC Criteria: July 7, 2015
• First Posted (Estimate): July 8, 2015

Study Record Updates

• Last Update Posted (Actual): September 26, 2019
• Last Update Submitted That Met QC Criteria: September 24, 2019
• Last Verified: September 1, 2019

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Immune System Diseases; Autoimmune Diseases; Eye Diseases; Endocrine System Diseases; Exophthalmos; Orbital Diseases; Goiter; Hyperthyroidism; Thyroiditis, Autoimmune; Thyroid Diseases; Hashimoto Disease; Graves Disease; Thyroiditis
• Other Study ID Numbers: 62/17-2-2014

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No