The Role of FOXP3 Polymorphisms in Graves’ Disease with or without Ophthalmopathy in a Turkish Population

Fulya Yaylacıoğlu Tuncay; Kübra Serbest Ceylanoğlu; Sezen Güntekin Ergün; Berçin Tarlan; Onur Konuk

doi:10.4274/tjo.galenos.2024.37897

ABSTRACT

Objectives:

Forkhead box P3 (FOXP3) gene polymorphisms have been evaluated in many autoimmune diseases, including Graves’ disease (GD), in different populations. However, those polymorphisms have not been analyzed in GD or Graves’ ophthalmopathy (GO) in the Turkish population. In this study, we aimed to evaluate the frequency of FOXP3 polymorphisms in GD with or without ophthalmopathy in a Turkish population.

Materials and Methods:

The study included 100 patients with GO, 74 patients with GD without ophthalmopathy, and 100 age- and sex-matched healthy controls. In all study participants, rs3761547 (-3499 A/G), rs3761548 (-3279 C/A), and rs3761549 (-2383 C/T) single nucleotide polymorphisms (SNPs) were detected using the polymerase chain reaction-restriction fragment length polymorphism method. The chi-square test was used to evaluate genotype and allele frequencies. Odds ratios and 95% confidence intervals were calculated for genotype and allele risks.

Results:

In the patient group (including GD with or without ophthalmopathy), the rs3761548 AC and AA genotype and rs3761549 CT genotype were significantly more frequent than in the control group (all p<0.05). No genotypic and allelic differences were observed for rs3761547 between the patient and control groups (all p>0.05). There was no statistically significant difference between the GO and GD without ophthalmopathy groups concerning the allele and genotype frequencies of all three FOXP3 SNPs (all p>0.05).

Conclusion:

The AC and AA genotypes of rs3761548 (-3279) and CT genotype of rs3761549 (-2383 C/T) were shown to be possible risk factors for GD development in the Turkish population. However, none of the three SNPs was shown to be associated with the development of GO in patients with GD.

Keywords:

Forkhead box P3, Graves’ disease, Graves’ ophthalmopathy, single nucleotide polymorphisms

Introduction

Graves’ disease (GD) is an autoimmune disorder that causes diffuse enlargement of the thyroid gland and hyperthyroidism with elevated thyroid-specific autoantibody levels. GD is more common in women, and it usually occurs between the ages of 20 and 40 years.^1,2 Up to 50% of GD patients also develop Graves’ ophthalmopathy (GO), which is another autoimmune disease that affects the orbital structures.³ GO varies in clinical severity and is assessed according to the European Group on Graves’ Ophthalmopathy (EUGOGO) classification.³

Thyroid-stimulating hormone receptor (TSHR) was shown to be the main autoantigen in GO, as in GD.⁴ Both diseases have a complex pathogenesis involving interactions between genetic and environmental factors.⁵ Among the genetic factors, CTLA-4, TSHR, Tg, CD40, and PTPN22 polymorphisms and HLA class II gene variants were shown to be shared risk factors between GO and GD.⁶ However, one polymorphism in IL1A was found to favor GO development in GD compared to GD patients without GO.⁷ Additionally, another study in the Polish population showed that a VDR polymorphism may contribute to the development of GO.⁸ From a clinical point of view, it is essential to identify patients with higher risk of developing GO in the course of GD, and we still need reliable genetic risk factors to act upon.

The immune system is primarily under the control of regulatory T-cells (Tregs).⁹ Tregs were shown to be pivotal factors in the pathogenesis of human autoimmune diseases, including GD and GO.^10,11,12 The Forkhead box P3 (FOXP3) gene is located on the X chromosome, and its protein product FoxP3 is predominantly expressed in Tregs as a transcription factor. A deficiency of FoxP3 may impair the immunosuppressive effect of Tregs and lead to autoimmune diseases.^13,14 An association between FOXP3 polymorphisms and the development of GD has been reported in different populations.^{10,13,15,16,17,18,19,20,21,22} According to a recent meta-analysis of seven case-control studies, the FOXP3 polymorphism rs3761548 was associated with GD susceptibility in Asians, and rs3761549 was associated in both Asians and Caucasians.¹⁵ Despite several studies about the relationship between FOXP3 single-nucleotide polymorphisms (SNPs) and GD susceptibility, none have been conducted in the Turkish population. Additionally, only two studies with small numbers of GO patients investigated the relationship between FOXP3 SNPs and the risk of GO in GD patients.^13,17 Therefore, our study is the first to investigate three SNPs in the FOXP3 gene (-2383 C/T, -3279 C/A, and -3499 A/G) in Turkish GD patients. In addition, we evaluated whether any of those SNPs favor GO development in a larger GD patient population.

Materials and Methods

Results

Discussion

FoxP3 is predominantly expressed in CD34+ CD25 Tregs and plays a critical role in maintaining the suppressive function of Tregs.^13,14 Genetic variations in the FOXP3 gene play a role in the pathogenesis of GD by weakening the suppressive functions of Tregs and enhancing autoimmune responses.^15,16,20 Although the relationship between FOXP3 SNPs and GD has been demonstrated in several studies in different populations, few studies to date have compared the frequency of FOXP3 SNPs between GD patients with and without orbitopathy.^13,17 This is the first study that investigated the relationship between three common polymorphisms in the FOXP3 gene (rs3761549 [-2383 C/T], rs3761548 [-3279 G/T], and rs3761547 [-3499 T/C]) and GD in a Turkish population. The results showed that the AC and AA genotypes of -3279 and the CT genotype of -2383 are possible risk factors for GD. However, the development of GO in GD patients could not be associated with the investigated FOXP3 polymorphisms in our study population.

For polymorphism -3279, an association between genotypes AA and AC and autoimmune diseases such as systemic lupus erythematosus and vitiligo has been shown in the literature.^10,12 The association with GD varies in the literature according to ethnicity. Although there are genotypic differences, the -3279 polymorphism has been reported to be a risk factor for GD in the Asian population.^{10,13,17,19,21,22} It has been noted that the AC genotype of -3279 in the Kashmiri population, the AA and AC genotypes of -3279 in the Chinese Han population, and the AA genotype of -3279 in the female Southwest Chinese Han population pose a risk for GD.^13,19,22 In addition, a high frequency of the A allele was reported in patients with high thyroid-stimulating hormone (TSH) levels or low TSHR levels.²¹ Like many studies, the A allele was observed more frequently in the GD group in the current study.^13,17,21 There are no studies in the Caucasian population reporting risk factors for -3279 polymorphisms.^18,20 Similar to the Kashmiri and Polish populations, the genotype distribution did not significantly differ between males and females in our study population (Table 5).^17,20 However, in the Asian population, the genotype distribution was reported to be significantly different between males and females.^13,22

Polymorphism -2383 has been reported to increase GD susceptibility similarly to polymorphism -3279.^13,17,18,20 Bossowski et al.²⁰ reported that among Caucasians, the CT genotype of the -2383 polymorphism was more common in healthy females. Shehjar et al.¹⁷ reported that the TT genotype of the -2383 polymorphism was a risk factor for developing GD in the Kashmiri population, but there were no sex differences in allelic or genotypic frequency distribution. In another study conducted in the Chinese Han population, carriers of the TT genotype of -2383 had a higher free triiodothyronine level than those with the CC/CT genotypes, but there was no significant difference between GD and control groups regarding genotype frequencies.¹³ In our study, we found an association between the development of GD and the CT genotype of -2383, and the T allele was significantly more frequent in the study group. However, the genotype distribution did not differ significantly between males and females in our study population (Table 5). The differences in allelic and genotypic associations with GD in studies may be explained by ethnic differences.

For polymorphism -3449, we found no statistically significant difference between groups, consistent with the literature.^{10,13,17,20,21,22} Only one study in the literature reported that free triiodothyronine and thyroxine levels and the -3499 A/G polymorphism were associated with GD.²⁰ It can be surmised that -3499 is not associated with altered FOXP3 expression and does not affect Tregs functions.

In our study, none of the FOXP3 genotypes or alleles was found to be associated with GO despite the higher number of GO patients in our study population. Similarly, Zheng et al.¹³ and Shehjar et al.¹⁷ could not show any associations between FOXP3 SNPs and ophthalmopathy in GD in Asian populations. In the literature, Aydın et al.²⁴ found an association between the endothelin receptor type A (EDNRA) C+70G gene and the development of ophthalmopathy in GD patients. Another study reported that new SNPs in CD74 (AG genotype of rs2569103) increased the risk of developing GO by affecting adipocyte proliferation and differentiation.²⁵ There are still many unanswered questions about the risk of ophthalmopathy in GD. For a better understanding of ophthalmopathy development, both genetic and non-genetic factors should be evaluated.

Conclusion

This study is the first to explore the association between FOXP3 polymorphisms and GD with and without GO in a Turkish sample. We showed that the AC and AA genotypes of -3279 and the CT genotype of -2383 may be risk factors for GD development in our study population. However, we could not find any association between FOXP3 SNPs and GO development in GD. More extensive population studies or meta-analyses of available data may reveal the impact of FOXP3 polymorphisms on the risk of GO development in patients with GD.

Study Limitations

Our study has several limitations. First, the number of participants in each group was limited, which might have reduced the power of the research and prevented us from showing significant differences between subgroups, such as gender and the presence of orbitopathy. Second, the study included only a small proportion of the GD patients in Türkiye. Therefore, whether our results could be generalized to the Turkish population is unclear. Third, we could not do a haplotype analysis due to the limited size of the study population. Fourth, our study was not longitudinal, and we could not control the other GO-related factors in study groups that might confound the risk of GO development in GD patients.

References

Wémeau JL, Klein M, Sadoul JL, Briet C, Vélayoudom-Céphise FL. Graves’ disease: Introduction, epidemiology, endogenous and environmental pathogenic factors. Ann Endocrinol (Paris). 2018;79:599-607.

Falgarone G, Heshmati HM, Cohen R, Reach G. Mechanisms in endocrinology. Role of emotional stress in the pathophysiology of Graves’ disease. Eur J Endocrinol. 2012;168:13-18.

Bartalena L, Baldeschi L, Dickinson AJ, Eckstein A, Kendall-Taylor P, Marcocci C, Mourits MP, Perros P, Boboridis K, Boschi A, Currò N, Daumerie C, Kahaly GJ, Krassas G, Lane CM, Lazarus JH, Marinò M, Nardi M, Neoh C, Orgiazzi J, Pearce S, Pinchera A, Pitz S, Salvi M, Sivelli P, Stahl M, von Arx G, Wiersinga WM. Consensus statement of the European group on Graves’ orbitopathy (EUGOGO) on management of Graves’ orbitopathy. Thyroid. 2008;18:333-346.

Bahn RS. Current Insights into the Pathogenesis of Graves’ Ophthalmopathy. Horm Metab Res. 2015;47:773-778.

Burch HB, Cooper DS. Management of Graves Disease: A Review. JAMA. 2015;314:2544-2554.

Lee HJ, Stefan-Lifshitz M, Li CW, Tomer Y. Genetics and epigenetics of autoimmune thyroid diseases: Translational implications. Best Pract Res Clin Endocrinol Metab. 2023;37:101661.

Wong KH, Rong SS, Chong KK, Young AL, Pang CP, Chen LJ. Genetic Associations of Interleukin-related Genes with Graves’ Ophthalmopathy: a Systematic Review and Meta-analysis. Sci Rep. 2015;5:16672.

Maciejewski A, Kowalczyk MJ, Herman W, Czyżyk A, Kowalska M, Żaba R, Łącka K. Vitamin D Receptor Gene Polymorphisms and Autoimmune Thyroiditis: Are They Associated with Disease Occurrence and Its Features? Biomed Res Int. 2019;2019:8197580.

Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol. 2004;22:531-562.

Yu M, Tan X, Huang Y. Foxp-3 variants are associated with susceptibility to Graves’ disease in Chinese population. Eur J Inflamm. 2017;15:113-119.

Saitoh O, Nagayama Y. Regulation of Graves’ hyperthyroidism with naturally occurring CD4+CD25+ regulatory T cells in a mouse model. Endocrinology. 2006;147:2417-2422.

Grant CR, Liberal R, Mieli-Vergani G, Vergani D, Longhi MS. Regulatory T-cells in autoimmune diseases: challenges, controversies and--yet--unanswered questions. Autoimmun Rev. 2015;14:105-116.

Zheng L, Wang X, Xu L, Wang N, Cai P, Liang T, Hu L. Foxp3 gene polymorphisms and haplotypes associate with susceptibility of Graves’ disease in Chinese Han population. Int Immunopharmacol. 2015;25:425-431.

Georgiev P, Charbonnier LM, Chatila TA. Regulatory T Cells: the Many Faces of Foxp3. J Clin Immunol. 2019;39:623-640.

Tan G, Wang X, Zheng G, Du J, Zhou F, Liang Z, Wei W, Yu H. Meta-analysis reveals significant association between FOXP3 polymorphisms and susceptibility to Graves’ disease. J Int Med Res. 2021;49:3000605211004199.

Li HN, Li XR, Du YY, Yang ZF, Lv ZT. The Association Between FoxP3 Polymorphisms and Risk of Graves’ Disease: A Systematic Review and Meta-Analysis of Observational Studies. Front Endocrinol (Lausanne). 2020;11:392.

Shehjar F, Afroze D, Misgar RA, Malik SA, Laway BA. Association of FoxP3 promoter polymorphisms with the risk of Graves’ disease in ethnic Kashmiri population. Gene. 2018;672:88-92.

Owen CJ, Eden JA, Jennings CE, Wilson V, Cheetham TD, Pearce SH. Genetic association studies of the FOXP3 gene in Graves’ disease and autoimmune Addison’s disease in the United Kingdom population. J Mol Endocrinol. 2006;37:97-104.

Fathima N, Narne P, Ishaq M. Association and gene-gene interaction analyses for polymorphic variants in CTLA-4 and FOXP3 genes: role in susceptibility to autoimmune thyroid disease. Endocrine. 2019;64:591-604.

Bossowski A, Borysewicz-Sańczyk H, Wawrusiewicz-Kurylonek N, Zasim A, Szalecki M, Wikiera B, Barg E, Myśliwiec M, Kucharska A, Bossowska A, Gościk J, Ziora K, Górska M, Krętowski A. Analysis of chosen polymorphisms in FoxP3 gene in children and adolescents with autoimmune thyroid diseases. Autoimmunity. 2014;47:395-400.

Inoue N, Watanabe M, Morita M, Tomizawa R, Akamizu T, Tatsumi K, Hidaka Y, Iwatani Y. Association of functional polymorphisms related to the transcriptional level of FOXP3 with prognosis of autoimmune thyroid diseases. Clin Exp Immunol. 2010;162:402-406.

Tan G, Zheng G, Li J, Zhu Y, Liang Z, Li H, Yu H, Wang X. Association of genetic variations in FoxP3 gene with Graves’ disease in a Southwest Chinese Han population. Immun Inflamm Dis. 2023;11:e1046.

Bahn Chair RS, Burch HB, Cooper DS, Garber JR, Greenlee MC, Klein I, Laurberg P, McDougall IR, Montori VM, Rivkees SA, Ross DS, Sosa JA, Stan MN; American Thyroid Association; American Association of Clinical Endocrinologists. Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists. Thyroid. 2011;21:593-646. Erratum in: Thyroid. 2011;21:1169. Erratum in: Thyroid. 2012;22:1195.

Aydın AF, Develi-İş S, Doğru-Abbasoğlu S, Vural P, Ozderya A, Karadağ B, Uysal M. Polymorphisms of endothelin 1 (G5665T and T-1370G) and endothelin receptor type A (C+70G and G-231A) in Graves’ disease. Int Immunopharmacol. 2014;18:198-202.

Liu YH, Shen CY, Tsai FJ. Association of variant on the promoter of cluster of differentiation 74 in graves disease and graves ophthalmopathy. Biosci Rep. 2020;40:BSR20202072.