1. Introduction
Loss of immune tolerance to tissue-specific antigenic peptides underlies autoimmune diseases and leads to an immune response directed against the body’s own cells. Complex immune mechanisms, including immune system dysfunction, are involved in the pathogenesis of autoimmune diseases [
1]. Common chronic autoimmune endocrine disorders in children include autoimmune thyroid diseases (AITDs), such as Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) [
2,
3]. The mechanisms leading to the development of these diseases remain unknown; however, scientific reports indicate that besides environmental factors, genetic background plays a significant role [
3,
4,
5]. The genetic predisposition to AITDs has been attributed to approximately 70% of disease risk, while environmental factors are believed to trigger the disease in genetically susceptible individuals [
3]. A number of genetic determinants of AITDs have already been established through candidate gene studies; however, the contribution of particular genes is not significant, and it is possible that polymorphisms play a role in multiple genes [
3].
Single-nucleotide polymorphisms (SNPs) are the simplest and most common form of DNA variation occurring throughout the genome. They can be responsible for variations among individuals, the evolution of the genome, familial traits, or inter-individual differences in responses to drugs and susceptibility to diseases. Identifying gene variants and analyzing their effects can lead to a better understanding of their impact on gene function and individual health [
6,
7]. This knowledge can provide a starting point for the development of new, useful markers for disease detection and therapeutic approaches.
In our previous studies, we demonstrated that some polymorphisms of the genes for IL2RA, FAIM2, IFIH1, PADI4, or CTLA-4 appeared more frequently in children and adolescents with autoimmune diseases such as type 1 diabetes (T1D) and AITDs, which may be related to the occurrence and course of the disease [
8,
9]. According to the literature, there are numerous other factors involved in immune mechanisms which may potentially affect the development of autoimmune diseases. These include Il-7 and its receptor Il7R, CD226, CAPSL, and CLEC16A, which appear to be attractive candidates for further studies to explore the pathogenic mechanisms and potential therapeutic options for autoimmune diseases.
The cytokine IL7 and its receptor, IL7R, are essential for T and B cell development, as well as the differentiation and survival of naive T cells and the generation and maintenance of memory T cells [
10,
11]. Stimulation of the receptor for Interleukin 7 (IL7R) has been shown to play an important role in the development and progression of autoimmune diseases [
12,
13,
14,
15]. The IL7R gene has been identified as one of the key genes and pathways associated with the development of Hashimoto’s thyroiditis [
16]. Furthermore, recent studies suggest that effects on the IL7/IL7R pathway may be important in the treatment of autoimmune diseases [
17,
18]. The IL7R is a heterodimer consisting of a common gamma chain (encoded by IL2RG) and a specific alpha chain (encoded by IL7R). Whereas the gamma chain is produced by most hemopoietic cells and is shared by the receptors of several cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21), the alpha chain is almost exclusively produced by lymphoid lineage cells and is required for the development and maintenance of the immune system [
19].
Adjacent to the IL7R gene, another gene is located on chromosome 5p13–CAPSL (Calcyphosine-like, also known as Q8WWF8). Studies indicate a link between CAPSL and the development of autoimmune diseases such as type 1 diabetes [
19]. The function of CAPSL is still unknown; however, it was established that it contains two calcium-binding motifs (EF-hands) also found in a superfamily of calcium sensors and calcium signal modulators [
19].
CD226 (also known as DNAM-1) is a membrane protein involved in cell adhesion, activation, and differentiation that has been shown to be expressed on the surface of immune cells including natural killer (NK) cells, CD4+ and CD8+ T cells, monocytes, platelets, and B18 cells. Polymorphisms in the gene encoding its molecule (located at chromosome 18q22.3) have been linked to a risk factor for autoimmune diseases like T1D and Graves’ disease [
20,
21,
22,
23,
24]. The possibility of CD226 affecting the treatment of autoimmune diseases has also been suggested [
25].
CLEC16A (C-Type Lectin Domain family 16A, previously known as KIAA0350) at chromosome 16p13 has been proposed as a gene region that may play a role as a susceptibility locus for autoimmune disease. Some SNPs within the CLEC16A gene have been shown to be associated with several autoimmune diseases, such as T1D, primary adrenal insufficiency, juvenile idiopathic arthritis, celiac disease, and AITDs, among others [
26,
27,
28]. Thus far, little is known about the detailed function of the CLEC16A gene product. It is highly expressed in B lymphocytes, NK cells, and dendritic cells (DC), and is thought to be involved in providing a signal for immune tolerance [
29].
The aim of this study was to assess the prevalence of selected single-nucleotide polymorphisms (SNPs) of Il7R, CD226, CAPSL, and CLEC16A genes in children and adolescents with autoimmune thyroid diseases, and in the control group. We compared these data in an attempt to demonstrate which of the studied SNPs may play a role in susceptibility to disease or show a protective effect against autoimmune thyroid disease in children and adolescents.
2. Results
We included in the study 56 patients with HT (mean age, 15.2 ± 2.2 years), 124 patients with GD (mean age, 16.5 ± 2 years), and 156 healthy, euthyroid volunteers (mean age, 16.3 ± 3 years) of comparable age, weight, and height as controls. The characteristics of the study group are presented in
Table 1.
The differences between AITD patients and a control group in the analyzed SNPs (rs3194051 and rs6897932 for the IL7R gene, rs763361 for the CD226 gene, and rs725613 for the CLEC16A gene) are presented in
Table 2 and
Table 3.
In our study, we observed a higher frequency of the C allele at the rs6897932 SNP of the IL7R gene in GD children in comparison to controls [
p = 0.03, odds ratio (OR) 1.52 and 95% confidence interval 1.04–2.24] (
Table 2). Furthermore, the C allele occurred more frequently in GD girls than in controls [
p = 0.017, odds ratio (OR) 1.82 and 95% confidence interval 1.11–3.00]) (
Table 4) and in HT boys than in the control group [
p = 0.017, odds ratio (OR) 6.58 and 95% confidence interval 1.30–160.93] (
Table 5). Moreover, the C/T genotype at the rs6897932 SNP of the IL7R gene was statistically significantly less frequent in all GD patients [
p = 0.007, with an odds ratio (OR) of 0.51 and 95% confidence interval of 0.30–0.83] (
Table 2). The data are presented in
Figure 1.
The study revealed the C/T genotype at the CAPSL locus (rs1010601) in HT boys to be statistically significantly less frequent in comparison to the control group [
p = 0.013, with an odds ratio (OR) of 0.09 and 95% confidence interval of 0.003–0.623] (
Table 6 and
Figure 2).
4. Materials and Methods
4.1. Patients
Our study was conducted on a group of 56 HT patients and 124 GD patients recruited from the Endocrinology Outpatient Clinic in Bialystok and the Department of Human Pathology in Adulthood and Childhood, University of Messina. Diagnosis of AITDs was based on the Polish Endocrinology Association guidelines, which correspond with the guidelines of the European Society for Pediatric Endocrinology. Inclusion in the study was determined by medical history, physical examination, laboratory tests, and ultrasound investigations. Children with HT showed clinical and biochemical symptoms of hypothyroidism and were positive for anti-TPO and/or anti-TG autoantibodies. Patients with GD presented large goiter, hyperthyroidism in laboratory tests, and positive thyrotropin receptor antibodies (TR-Ab). All patients had no other diagnosed autoimmune diseases. HT patients were treated with L-thyroxine at a dose of 1 mcg/kg/day orally and GD children received methimazole at a dose of 0.3–1.0 mg/kg/day together with propranolol (0.5–1.0 mg/kg/day) orally. The control group consisted of 156 healthy, euthyroid volunteers with no history of HT or GD and no thyroid autoantibodies. Informed consent was given by all parents of the patients and controls as well as all children over 16 years old prior to study inclusion. The study protocol was accepted by the Local Ethical Committee at the Medical University of Bialystok and adheres to the Declaration of Helsinki.
4.2. Blood Analysis
Blood samples for analysis were collected from the basilic vein, fasting, in the morning. The serum concentrations of thyrotropin (TSH), free thyroxine (fT4), and free triiodothyronine (fT3) were evaluated on electrochemiluminescence “ECLIA” with the Cobas E411 analyzer (Roche Diagnostics). Normal values for TSH were 0.28–4.3 (μIU/L), 1.1–1.7 ng/dL for fT4, and 2.3–5.0 pg/mL for fT3. Antibodies against TSH-Receptor (TR-Ab), Thyroid Peroxidase (TPO), and Thyroglobulin (TG) were determined using ECLIA with Modular Analytics E170 analyzer (Roche Diagnostics). Positive titer for TR-Ab were >1.75 U/L, >34 IU/mL for anti-TPO-Ab, and >115 IU/mL for anti-TG-Ab.
4.3. DNA Extraction
DNA was extracted with a classical salting-out method from the blood leukocytes. All study subjects were genotyped for SNPs at the loci rs3194051 and rs6897932 for the IL7R gene, rs763361 for the CD226 gene, rs1010601 for the CAPSL gene, and rs725613 for the CLEC16A gene. TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA) was used for all genotyping. For this, polymorphisms fluorogenic TaqMan probes were used. Reactions were performed in a 7900HT fast real-time PCR system (Applied Biosystems) according to the following conditions: 10 min at 95 C for starting AmpliTaq Gold activity, 40 cycles at 95 C for 15 s, and 60 C for 1 min. As a negative control served a sample without a template. It was helpful to detect any false positive signal caused by contamination. All SNPs were analyzed in duplicates.
4.4. Statistical Analysis
The median unbiased estimator (mid-p) of the odds ratio, the exact confidence interval, and the associated
p-value obtained with the mid-p method were used to determine any association between genotype or allele occurrence and a patient’s status [
39]. Either parametric or non-parametric methods, according to the normality and homogeneity of variance assumptions, were used to assess whether there are statistically significant differences between groups defined by genotypes and quantitative features. The false discovery rate
p-value adjustment method was applied due to the issue of multiple testing during the post hoc analysis [
40]. As proposed in [
41], measure D’ of linkage disequilibrium was used. For all calculations, a
p-value of <0.05 was considered to be significant. The R software version 4.3.1 (16 June 2023) (Vienna, Austria) environment was utilized for all calculations [
42]. Statistical power calculation with respect to the total sample size was calculated with the use of G
*Power ver. 3.1.9.6 software [
43]. Cohen’s w was applied as a measure of effect size. The Hardy–Weinberg Equilibrium was checked with the utilities of the genetics package [
44].