Comprehensive analysis of chemokine gene polymorphisms in Korean children with autoimmune thyroid disease

Chemokines are chemotactic cytokines that can cause directed migration of leukocytes. The aim of this study was to examine differences in single nucleotide polymorphisms (SNP) of chemokine in AITD patients compared to normal controls. A total of 86 Korean pediatric patients were included in the patient group and 183 adults were included in the normal control group. To compare influences of several chemokine gene polymorphisms, 25 SNPs in 16 chemokine genes were analyzed. Genotype frequencies of CCL11(rs3744508)AA(OR = 6.9) and CCR2(rs1799864)AA(OR = 3.8) were higher in the AITD patients than in the controls, whereas CCL17(rs223828)CC was lower in the AITD patients than in the controls(OR = 0.4). In comparison between Graves' disease (GD) patients and controls, genotype frequency of CCL17(rs223828)CC(OR = 0.4) was lower in the GD group, whereas those of CCR2(rs1799864)AA(OR = 4.8) were higher in the GD group. The genotype frequency of CCL11(rs3744508)AA(OR = 11.3) was higher in Hashimoto's thyroiditis (HT) patients, whereas that of CXCL8(rs2227306)CC(OR = 0.4) was lower in HT patients. Polymorphisms of CCL11(rs3744508), CCL17(rs223828), and CCR2(rs1799864) might be associated with AITD, with CCL17(rs223828), CCR2(rs1799864) and CXCR2(rs2230054, rs1126579) affecting GD and CCL11(rs3744508) and CXCL8(rs2227306) affecting HT in Korean children.


Materials and methods
Subjects.The present study was conducted on 86 patients diagnosed with AITD [36 with HT and 50 with GD (intractable GD, n = 30) (TAO, n = 24; non-TAO, n = 26)] who were treated at the Pediatric Endocrine Clinics of Seoul St. Mary's Hospital between March 2009 and August 2021.The age of patients in the study was 13.2 ± 3.3 years at enrollment, and 11.3 ± 3.2 years at diagnosis of AITD (Table 1).
For the control group, a total of 183 healthy Korean adults (52.5% females and 47.5% males; average age: 29.9 ± 3.7 years) without a history of AITD comprising staff and students from the College of Medicine at the Catholic University of Korea were included.All participants provided an informed consent for a genetic study.This study was approved by the Institutional Review Board (IRB) of the Catholic University of Korea (IRB Number: KC09FISI0042, MC13SISI0126), Seoul, Korea, and the study was conducted in accordance with the Declaration of Helsinki.
GD was diagnosed based on clinical symptoms and biochemical confirmation of hyperthyroidism, including diagnosis of goiter, elevated radioactive iodine uptake, antibodies against the thyroid-stimulating hormone (TSH) receptor, and elevated thyroid hormone levels.Patients with other forms of autoimmune diseases, hematologic diseases, or endocrine diseases were excluded.TAO was diagnosed based on the presence of typical clinical features.It was classified according to the system recommended by the American Thyroid Association (ATA) Committee 18 .Patients with no symptoms or the lid lag sign only were included in the non-TAO group, whereas those with soft tissue changes, proptosis, extraocular muscle dysfunction, or the latter two were considered to have an eye disease 19 .In GD, a remission was defined as consistent with improvement of clinical features and restoration of euthyroidism or induction of hypothyroidism after antithyroid drug (ATD) therapy.We defined intractable GD as hyperthyroidism persistent over two years of ATD therapy, relapse after ATD withdrawal, or treatment with ATD for at least 5 years [20][21][22] .HT was diagnosed when at least three of the following criteria established by Fisher et al. 23 were met: (1) goiter, diffuse goiter, and decreased radionuclide uptake during thyroid scan, (2) circulating thyroglobulin or microsomal autoantibodies, and (3) hormonal evidence of hypothyroidism.

Discussion
In this study, we observed significant differences in the frequencies of SNPs CCL11(rs3744508), CCL17(rs223828), and CCR2(rs1799864) were observed between the control group and AITD in Korean children.The AITD group had a higher T allele frequency in CCL17(rs223828), higher genotype frequencies in CCL11(rs3744508) AA and CCR2(rs1799864)AA, and a lower frequency in CCL17(rs223828)CC compared to controls.Upon dividing AITD into GD and HT, we found significant differences in CCL17(rs223828), CCR2(rs1799864), and CXCR2(rs2230054, rs1126579) between the control group and the GD group.Among GD patients, frequencies of CCR2(rs1799864)AA, CXCR2(rs2230054)CC, and CXCR2(rs1126579)TT were higher, and CCL17(rs223828) CC was lower compared to controls.In HT patients, CCL11(rs3744508)AA was higher, and CXCL8(rs2227306) CC was lower than controls.Associations between chemokine SNPs and immune-related diseases, including AITD, have been previously documented.The T allele in CCL17(rs223828) was associated with increased serum CCL17 levels as well as increased coronary artery disease risk in a Chinese Han Population 29 .In relation to CCL17, Aso et al. 30  www.nature.com/scientificreports/with reports of higher levels of CCL17 and CXCL10 in serum in GD patients accompanied by type 1 diabetes.SNPs such as CCL11(rs1129844) have also been connected with elevated plasma chemokine levels in conditions like Fibromyalgia 31 .In the population of India, a significant association was found between SNPs CCR2 (rs1799864A) and Japanese encephalitis 32 , an observation supported by the overexpression of CCL2 (a ligand that can bind to CCR2) in GD 33 .Even though no direct connections have been reported between cxcr2 polymorphism and AITD, research has uncovered associations with other medical conditions.For instance, in China, the CXCR2 (rs2230054) CC genotype occurred more frequently in patients with thoracic aortic aneurysm 34 .Additionally, individuals carrying the CXCR2 rs1126579 TT genotype showed a significant increase in the likelihood of HCV spontaneous clearance 35 .Early studies focusing on chemokines of HT have revealed the role of CXCL8 36 .Weetman et al. 37 demonstrated that thyroid cells express CXCL8 upon stimulation with inflammatory cytokines such as IFN-r, TNF-α, and IL-1.Interestingly, the CC genotype of CXCL8(rs2227306) was found to be associated with susceptibility to sepsis in males 38 , indicating a broader impact of these polymorphisms on immune function and disease susceptibility.
In addition to the chemokine SNPs significantly identified in our study, various other chemokine SNPs have been associated with GD or HT, underlining the complexity of the relationship between these genetic variations and AITD.Several chemokines, including CCL3, CCL4, CCL21, CXCL9, CXCL10, and CXCL11, have been reported to link specifically to GD and HT 37,[39][40][41][42][43][44][45] .In the early stage of GD, relationships are seen between chemokines and immune cells.CXCL10, made by follicular epithelial cells in the thyroid, attracts specific immune cells called CXCR3-expressing Th1 cells, leading to inflammation 41 .Research has shown that people with GD have more CXCL10 in their blood than people of the same age and gender without GD 42 .This same pattern has been found with other similar proteins, CXCL9 and CXCL11, where higher levels were found in the blood of people with GD compared to those without the condition 43 .The functional dynamics of chemokines in AITD have also been explored.Other research has examined the interaction of chemokines and other cytokines in primary thyrocyte cultures from GD patients, discovering that PPAR-α ligands can inhibit the secretion of CXCL9, CXCL10, and CXCL11 in a dose-dependent manner 44,45 .This finding provides potential insights into therapeutic pathways.Furthermore, Garcia-Lopez et al. identified increased expression levels of CXCL9 and CXCL10 in the thyroid of HT patients, thus revealing a specific association of CXCL9 and CXCL10 with HT 46 .These observations collectively illuminate the multifaceted role of chemokines in the pathogenesis of AITD, and underscore the need for continued investigation into these complex interactions.This study uncovered statistically significant differences in the frequencies of SNPs in CCL17, CCR2, and CXCR2 between the GD group and the control group.Additionally, we identified significant differences in the frequencies of SNPs of CCL11 and CXCL8 between the HT group and the control group.These findings not only confirm the association between the CXCL8 gene polymorphism and HT but also suggest that CCL11 might be a potential contributing factor to HT.In practical terms, these insights could lead to novel therapeutic avenues.Targeting chemokines through antagonists, such as CCR4, CCR5, and CXCR4, might present a new strategy for treating AITD and other related conditions.This emphasizes the importance of understanding these genetic variations, as they could provide key information for the development of future treatments.
This study has some limitations.First, the power of statistics is inevitably low because of small number of patients.Second, we did not investigate how the specific genetic variations, or SNPs, we identified influence the levels of chemokines in the patient's blood.Understanding this relationship could offer useful insights for predicting the disease.Future research should focus on exploring how these SNPs affect serum levels, as this could provide a more comprehensive understanding of the underlying mechanisms in AITD.In conclusion, this study identified six SNPs in five genes (CCL11, CCL17, CCR2, CXCR2, and CXCR8) that may be linked to an increased risk of AITD, GD, and HT in Korean pediatric patients.These findings point to a potential genetic relationship between these specific SNPs and the development of AITD, offering a new perspective on the underlying mechanisms of the disease.This requires larger sample sizes and possibly the inclusion of samples from diverse regions, to build a more comprehensive and definitive understanding of these genetic influences on AITD.

Table 2 .
Gene information.§ SNP information referred from dbSNP (https:// www.ncbi.nlm.nih.gov/ snp).*Span: The distance between forward and reverse primers on target sequence.Universal primer: The universal sequences were not matched with any genomic DNA sequences.All the primers include a universal sequence at individual 5′-end.

Table 5 .
GD related genotype and allele.