Autoimmune thyroid diseases (AITDs) are autoimmune disorders affecting thyroid gland. Hyperthyroidism, Graves’ disease (GD), and hypothyroidism, Hashimoto’s thyroiditis (HT), are the most common forms of AITDs, GD presents clinically as hyperthyroidism due to the production of thyroid-stimulating autoantibodies. [1]. These autoimmune disorders affect approximately 2% to 5% of the global population and notably high prevalence observed in females (5%–15%) compared to males (1%–5%) [2]. GD represent an autoimmune thyroid gland disorder, recognized by the presence of circulating immunoglobulins directed against thyroid stimulating hormone receptor (TSH-R). These autoantibodies cause excessive thyroid hormone production and the clinical manifestation through persistent activation of the receptors. [3]. Clinically, GD is presented by the occurrence of circulating antithyroid antibodies (ATA), thyrotoxicosis along with the detection of the infiltration of autoreactive lymphocytes into the thyroid gland, resulting an underlying autoimmune reaction. [4]. GD related systemic symptoms represent anxiety, fatigue, weakness, palpitations, diaphoresis, weight loss, and nervousness, it may involve extrathyroidal manifestations including autoimmune-related complications such as pretibial myxedema, thyroid-associated orbitopathy (TAO). [5] Cytokines are essential mediators in immune regulation and thyroid follicular cell modulation, playing a vital role in contributing to both the initiation and progression of inflammatory responses in GD. The most important pro-inflammatory cytokines like gamma interferon (INF-γ), is secreted by infiltrating immune cells and thyroid follicular cells within the inflamed gland [6]. In addition to inflammatory responses, these pro-inflammatory cytokines have a crucial role in orchestrating the recruitment and activation of immune effector cells to sites of tissue injury, augmenting the autoimmune reaction in GD [7]. Epigenetics serves as a crucial mediator in integrating genetic and environmental influences,      offering       insights     into   gene expression regulation without modulating the DNA sequence. The most significant epigenetic mechanisms implicated in the pathogenesis of AITD include X-chromosome inactivation, methylation of DNA, non-coding RNAs and histone modifications [8]. Aberrant DNA methylation affects both immune-related and thyroid-specific genes in GD, with hypomethylation of immune-associated genes promoting the production of thyroid-stimulating antibodies. Although studies on DNA methylation in AITD are limited and heterogeneous, they collectively support a significant role for abnormal methylation patterns in disease pathogenesis. [9]. .The primary objective of current investigation was to comprehensively evaluate the serum concentration of pro-inflammatory cytokin INF-γ, In addition to immunophenotyping, the study also aimed to investigate the epigenetic regulation of these INF-γ by assessing the DNA methylation status within the promoter regions of their corresponding gene in patients diagnosed with GD in Diwaniya City/ Iraq.

Table 1: Primers Utilized Current This Study

Sample Collection

The study population consists of two groups:42 patients clinically diagnosed with GD and 38 healthy control individuals, all recruited from private endocrinology and oncology clinics in Diwaniya city between November 2023 and May 2024. Patients were diagnosed based on clinical evaluations performed by endocrinologist and oncologist and further confirmed through laboratory parameters, including elevated levels of Anti-Thyroid Peroxidase (TPO) antibodies and suppressed Thyroid Stimulating Hormone (TSH). 

Evaluation of TPO and TSH

Blood samples collected from GD patients were first centrifuged to separate the serum from cellular components. The serum was then aliquoted and stored at -20°C until analysis to maintain sample integrity. Prior to testing, the samples were thawed to room temperature and gently mixed to ensure homogeneity.) used in the present study to evaluate TPO and TSH using VIDUS® T4 and VIDUS® Anti-TPO kits (BIOMRIEUX/ France) by VIDUS system (BIOMRIEUX/ France).

Evaluation of Interferon- γ (IFN-γ) levels

ELISA method was performed for determination of INF -γ levels in serum samples. this kid was provided by used Interleukin- INF- γ ELISA-Kit (Elabscience. USA).

Extraction of the Genomic DNA

Blood samples genomic DNA is harvested through using of g-SYAN DNA (Frozen Blood) kit manufactured by Geneaid- USA, and conducted as manufacture

Methylation Percentage Quantification and PCR Condition

Methyl specific qPCR (MSP) method used in the present study for Quantification of methylation percentage in promoters of the interleukin relative to internal control (GAPDH) after Bisulfite conversion of the extracted DNA using MethylEdge^TM Bisulfite Conversion System kit (Promega/ USA). Master mix of qPCR is conducted by using Master Mix kit (Go Taq® qPCR) depending on SYBER-Green identification of amplified gene which designed through data base of NCBI Gene Bank and MethPrimer online software (www.urogene.org/methprimer/index1.html). Primers for each gene included two sets of primers, M for methylated cites and U for unmethylated cites in the promotor,table (1). Promotor sequences obtained from Eukaryotes Promoter Database (EPD), https://epd.expasy.org/epd/ .Primers obtained from (ScientificResearcher. Co. Ltd. Iraq). Methylation percentage calculated as the following equation:

Demographic Characterization

The present showed that the patient group consisted of 31(73.80%) females and 11(26.20%) males totaling 42 participants; male to female ratio is 0.35/1. In the control group, there were 23 (60.52%) females and 15 (39.48%) males, resulting in a total of 38 participants, Table 2. Statistical analysis confirming the difference between patients and controls observed non-significant.

Table 2: Sex Distribution of Graves’ Disease and Healthy Control Group

*= Pearson Chi-Square Test With 0.05 Significance Level, ** Non- Significant

Sex distribution was a key demographic variable assessed in GD patients, given the well-documented sex-related differences in autoimmune thyroid diseases, particularly the higher prevalence observed in females. Autoimmune conditions such as GD are known to exhibit a strong gender bias, often attributed to a combination of hormonal, genetic, and immune regulatory factors that differ between males and females [10]. In line with epidemiological trends reported worldwide, including Iraq, the patient cohorts demonstrated a pronounced female predominance, consistent with the broader understanding of GD being more commonly diagnosed in women, the present results aligned with local and international studies that demonstrated high predominance of female in GD [11-15].

The demographic characteristics of the study classified age into five groups, results revealed a comparative distribution between the patient and control groups as showed in Table 3. Among the 80 participants, the patient group (n = 42) comprised 6 individuals were <20 years, 13 in the 20–29 years category, 8 in both 30–39 and 40–49 years categories, and 7 aged above 50 years. In contrast, the control group (n = 38) included 3, 10, 11, 5, and 9 individuals in the respective age categories. There is non-significant variation between patients and controls in age distribution as indicated by statistical analysis, (p = 0.624). Non-significant variation in age distribution between patient and control group supports the suitability of group matching which decrease confusing potential in the comparisons. In current study, the age distribution of GD patients aligns with local and universal established epidemiological patterns, which indicates that the situation may appear in a wide age limit [16-18]. The GD is widely identified to demonstrate the beginning of a bimodal or variable age in various population, usually with young adulthood and peaks seen in a middle age, although it can occur at any standard of living. Initially, this variability can reflect the dynamics of the immune system, hormonal effects and the difference in environmental trigger throughout life. [19,20].

Table 3: Age Distribution of Graves’ Disease and Healthy Control Group

Pearson Chi-Square Test With 0.05 Significance Level, NS = non-Significant

Evaluation of Thyroid Stimulating Hormone (TSH) and Thyroid Peroxidase Antibodies TPO

Our study showed, as indicated in table (4), that there is a significant difference between the patient and control groups in TSH and TPO levels. In the TSH measurement, the patient group exhibited a mean value of 0.462±0.398, In contrast, the control group, comprising 38 individuals, demonstrated a mean TSH level of 2.075±1.032, yielding a statistically significant, p-value of 0.001.

Table 4: Evaluation of TSH and TPO in Graves’ Disease and Healthy Control Group

Independent sample t-test with 0.05 Significance Level, S= Significant

Moreover, concerning TPO levels, the patient group again showed remarkable findings, with a mean of 159.39±101.74. This result also yielded a significant difference (p = 0.001). The control group, on the other hand, had a mean TPO level of 22.12±11.39, with a minimum of 3.07 and a maximum of 36.98. TSH suppression is a hallmark of hyperthyroidism, which is typically seen in GD. The significantly lower mean TSH levels in patients compared to controls suggest central downregulation of TSH due to elevated circulating thyroid hormones, a common pathophysiological mechanism in Graves’ disease [21]. On the other hand, anti-TPO antibodies are markers of autoimmune thyroiditis, their presence in elevated levels among Graves’ disease patients indicates underlying immune-mediated thyroid pathology, suggesting an overlap or coexistence of autoimmune mechanisms between the two conditions [22]. These findings align with established researches where TSH and anti- TPO antibody measurements are integral components of the diagnostic algorithm for autoimmune hyperthyroidism [23, 24].

Quantification of Interferon Gamma (INF-γ) Levels

The evaluation of INF-γ levels in the patient and control groups results is systematically organized in Table 5. The patient group demonstrated a mean INF- γ level of 432.49±53.22, while the control group exhibited a lower mean value of 401.58±64.08, Figure 1. Difference between the two groups was statistically significant, p-value 0.021. In GD, evaluation of IFN-γ levels underscores a crucial role for this cytokine in the immunopathology mechanisms in this disorder. IFN- γ, a hallmark cytokine of th1 cells, plays a central role in promoting cellular immunity and modifying antigen presentation, which includes effects on thyroid follicular cells that can affect autoimmune reactions [25]. Significant increased levels in IFN-γ in the patient group in current study supports the hypothesis that a Th1-polarized immune response may be included in the pathogenesis or perpetuation of the disease. Compared with other studies, current results agree with other studies indicating elevated IFN-γ in serum of patients with GD, conforming systemic immune activation [26-29].

Table 5: Levels of INF-γ Levels in Graves’ Disease and Healthy Control Group

Independent sample t-test with 0.05 Significance Level, S= Significant.

Figure 1: Mean of IFN- γ in Graves’ Disease Group and Control Group

Diagnostic Accuracy of IFN- γ

The diagnostic accuracy of IFN- γ was evaluated using a Receiver Operating Characteristic (ROC) curve analysis, with the aim of distinguishing between patients and healthy controls. Analysis conducted with a cut-off value for INF-γ at 438.44, Figure 2, Table 6. Individuals were categorized based on whether theirINF- γ levels fell below or above this threshold. Among the patient group, 19 individuals had INF-γ levels below the cut-off, compared to 26 in the control group, while 23 patients and 12 controls exhibited levels above the cut-off. The Area Under the Curve (AUC) was determined to be 0.638, with a 95% confidence interval (CI) ranging from 0.517 to 0.760, indicating poor diagnostic potential.

The sensitivity of the test, which measures the proportion of correctly identified patients, was notably low at 5.48%, whereas the specificity, representing the proportion of correctly identified controls, was 68.4%. These findings agree somewhat with those reported by Cheng et al., who highlighted the relevance of IFN-γ in modulating disease severity and thyroid-stimulating hormone receptor antibody (TSHR-Ab) titers in women with established GD, suggesting that while IFN-γ may play a role in disease pathophysiology or progression, its utility as a diagnostic tool remains limited [30]. On the other hand, a study by Rashad et al identified serum IFN-γ levels as sensitive and specific biomarkers for HD. suggesting that IFN-γ may play a crucial role in the pathogenesis and could serve as a reliable diagnostic biomarker of this condition [31]. This discrepancy underlines its role in GD, more complex, possibly reflects the severity of the disease rather than serving as a straight clinical tool.

Figure 2: ROC Curve of INF- γ Accuracy

DNA Methylation Percentage Quantification of INF-γ

The present study demonstrated variations between two groups methylation percentages in CpG sites of INF-γ promotor. In the GD group, the mean methylation percentage was recorded at 48.854, with a median value of 61.402 and an interquartile range (IQR) of 56.60. Conversely, the control group exhibited a higher mean methylation percentage of 58.67, with a median of 67.50 and a narrower IQR of 29.93. Statistical assessment indicated nonsignificant difference of the two groups, P-value 0.540, which exceeds the conventional threshold of 0.05 for statistical significance, table (7) and Figure (3).

The observed hypomethylation of the IFN-γ promoter in the studied group aligns with existing evidence that DNA methylation at CpG-rich regulatory regions has a critical effect in the lineage commitment and functional polarization of T-cells. Promoter hypomethylation is generally associated with increased chromatin accessibility and permissiveness for gene transcription, particularly in terminally differentiated effector T cells such as Th1 cells, which are characterized by high IFN-γ expression. The relatively lower mean methylation levels suggest a potential epigenetic priming or active state of the IFN-γ locus, which may reflect shifts in the composition or activation status of the Tcell pool. [32,33]. However, the lack of statistically significant differences between the groups indicates that, at the population level, the methylation profile of the IFN-γ promoter does not distinguish the studied cohort from controls. The finding of increased IQR range observed in IFN-γ promoter methylation suggests greater variability in methylation status which reflects variability in cellular composition, intrinsic immune activation states or environmental factors [34]. Rekha et al have shown a differential association of high and low producing alleles of IFN-γ with GD patients compared with controls [27], the study suggest that genetic and epigenetic factors may influence cytokine production and immune dysregulation. Collectively, our study the complexity of regulation in cytokine genes in autoimmune conditions, highlights the need to investigate both genetic and epigenetic mechanisms of immune cell function [35,36].

Figure 3: Box Plot of Median Comparison of DNA Methylation Percentages in CpG sites of INF-γ in Graves’ Disease and Healthy Control Group

Figure 4: ROC Curve of percentages of methylation in the promoter of IFN- γ

Table 6: Accuracy Characteristics of INF- γ

IFN- γ promoter methylation percentages Diagnostic Accuracy.

ROC curve conducted to velate the diagnostic accuracy of methylation percentages in the promoter region of studied cytokines. Results demonstrated that IL-17 ROC curve conducted at a methylation percentage cutoff value76.56. Individuals with methylation percentages above this threshold included 27 patients and 34 controls, while those with methylation percentages below the threshold consisted of 15 patients and 4 controls. The area under the ROC curve (AUC) was calculated to be 0.540, reflecting the overall poor ability of the methylation percentages as a biomarker. The sensitivity of the test, which indicates the proportion of correctly identified positive cases among patients, was found to be 0.333, whereas the specificity, representing the proportion of correctly identified negative cases among controls, was notably higher at 0.905, Table 8.

Table 7: Comparative Analysis INF-γ Promoter CpG Site Methylation Profiles in Graves’ Disease Patients Versus Healthy Controls

M. Whitney U-test; NS = nonsignificant at p>0.05, IQR = Interquartile Range

Table 8: Diagnostic Accuracy Characteristics of methylation percentages in the promoter of IFN- γ

Comparable to the findings for certain interleukin genes, the analysis of DNA methylation in the IFN-γ promoter did not yield results that clearly differentiate between groups. Additionally, there is a lack of existing data available for comparison, making it difficult to place these findings within a broader epigenetic or immunological context. The limited discriminative potential of the methylation percentages suggests that, at least in this setting, promoter methylation alone may not be sufficient to serve as a robust biomarker.

It was observed that INF-γ levels are significantly elevated in patients with Graves’ disease (GD) compared to healthy controls. Furthermore, analysis of the methylation status of the INF-γ gene promoter revealed a trend toward hypomethylation in GD patients, suggesting potential epigenetic modulation of gene expression. However, the observed difference in methylation levels between the two groups did not achieve statistical significance, possibly due to limitations such as sample size or variability within the study population. Nevertheless, the consistent pattern of hypomethylation in conjunction with increased INF-γ expression implies that DNA methylation within the INF-γ promoter region may play a regulatory role in the pathogenesis of Graves’ disease, warranting further investigation into its functional implications.

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