Endocrine Abstracts

Objectives: Graves’ Disease (GD) is an autoimmune disorder and it is a type of hyperthyroidism. As for its multifactorial etiopathogenesis, it involves various causative agents - first and foremost genetic, as well as environmental (i.e. smoking) and existential ones. Apparently, children may be the group most impacted by genetic influences. Moreover, given disparate clinical responses to antithyroid agents depending on affected patients’ age – finding novel GD pathogenesis factors, like epigenetics, seems to be justified. In literature, existence of DNA methylation (DNAm) pattern abnormalities in adult GD patients, as well as dynamic, reversible site-specific genome methylation changes in response to cigarette smoking and antithyroid therapy have also been shown. With this in mind, the aim of the research entailed characterization of whole blood genome DNAm pattern in non-smoking, newly diagnosed pediatric and adult patients with GD.

Methods and Results: In this two-centres prospective study, we enrolled a total of 11 hyperthyroid patients with GD diagnosed in line with ETA guidelines: 5 children (10-17 years, group 1; GR1) and 6 adults (34-52 years, GR2), respectively. We included healthy controls for children (HC; n = 5, 10-17 years) into research as well. All GD patients have not been treated with antithyroid agents prior to sampling and were negative towards smoking. In all patients we gathered whole blood frozen first. DNA was isolated using a Genomic DNA kit, this being followed by material quality assessment. Then, we prepared methyl-DNA libraries in line with the NEXTflex protocol with a bisulfite conversion and performed reduced representation bisulfite sequencing (RRBS, Illumina, HiSeq platform). Bioinformatic analysis served to find the ratio of non- and methylated genome cytosines (5-methylcytosine:cytosine ratio, 5MC:C) in differentially methylated regions (DMR), as well as qualitative analysis of principal components (PCA) and specific regions. The statistical-significance involved P-value and q-value for limiting false-positive results basing on false discovery rate (FDR) method. Primarily, a total of 21038092 (GR1 vs. HC) and 20720785 (GR1 vs. GR2) sites were analyzed. We noticed following total 5MC:C ratios’ ranges: 54,5-57,3% in GR1, 56,6-58,4% in GR2 and 57,3-59,9% in HC, respectively. Noteworthy, we found no significant differences in PCA, as well as in case of specific sites, promoters, genes and Graves’ loci post to FDR correction.

Conclusions: Newly diagnosed GD patients with negative history towards smoking present with insignificant differences in the whole-genome DNAm pattern regardless of age of onset.