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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
Sponsor:
Information provided by (Responsible Party):
Assimina Galli-Tsinopoulou, Aristotle University Of Thessaloniki

Tracking Information
First Submitted Date December 23, 2014
First Posted Date July 8, 2015
Last Update Posted Date September 26, 2019
Actual Study Start Date September 2014
Actual Primary Completion Date September 2016   (Final data collection date for primary outcome measure)
Current Primary Outcome Measures
 (submitted: September 24, 2019)
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).
Original Primary Outcome Measures
 (submitted: July 7, 2015)
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 ]
Change History
Current Secondary Outcome Measures
 (submitted: September 24, 2019)
  • 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
Original Secondary Outcome Measures
 (submitted: July 7, 2015)
  • 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 ]
Current Other Pre-specified Outcome Measures Not Provided
Original Other Pre-specified Outcome Measures Not Provided
 
Descriptive Information
Brief Title DNA Methylation and Autoimmune Thyroid Diseases
Official Title Study of DNA Methylation in Children and Adolescents With Autoimmune Thyroid Diseases
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 Type Observational
Study Design Observational Model: Case-Control
Time Perspective: Cross-Sectional
Target Follow-Up Duration Not Provided
Biospecimen Retention:   Samples With DNA
Description:
Blood samples will be collected and centrifuged and then leukocytes will be separated. DNA will then be isolated from peripheral leukocytes using the QIAamp DNA Blood Mini Kit.
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.
Condition
  • Hashimoto Thyroiditis
  • Graves Disease
Intervention Not Provided
Study Groups/Cohorts
  • 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
Publications *

*   Includes publications given by the data provider as well as publications identified by ClinicalTrials.gov Identifier (NCT Number) in Medline.
 
Recruitment Information
Recruitment Status Completed
Actual Enrollment
 (submitted: September 24, 2019)
110
Original Estimated Enrollment
 (submitted: July 7, 2015)
100
Actual Study Completion Date April 2018
Actual Primary Completion Date September 2016   (Final data collection date for primary outcome measure)
Eligibility Criteria

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
Sex/Gender
Sexes Eligible for Study: All
Ages 4 Years to 18 Years   (Child, Adult)
Accepts Healthy Volunteers Yes
Contacts Contact information is only displayed when the study is recruiting subjects
Listed Location Countries Greece
Removed Location Countries  
 
Administrative Information
NCT Number NCT02491567
Other Study ID Numbers 62/17-2-2014
Has Data Monitoring Committee No
U.S. FDA-regulated Product
Studies a U.S. FDA-regulated Drug Product: No
Studies a U.S. FDA-regulated Device Product: No
IPD Sharing Statement Not Provided
Responsible Party Assimina Galli-Tsinopoulou, Aristotle University Of Thessaloniki
Study Sponsor Aristotle University Of Thessaloniki
Collaborators Not Provided
Investigators
Principal Investigator: Assimina Galli-Tsinopoulou, Professor Medical School, Aristotle University of Thessaloniki
PRS Account Aristotle University Of Thessaloniki
Verification Date September 2019