DIABETES/METABOLISM RESEARCH AND REVIEWS R E S E A R C H A R T I C L EDiabetes Metab Res Rev 2011; 27: 867–871.Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/dmrr.1263

Evidence for the role of STAT4 as a generalautoimmunity locus in the Korean population

Yongsoo Park1∗

Hye-Soon Lee1

Youngjoo Park2

Dongsoo Min1

Seiwon Yang2

Dukhee Kim3

Boyoun Cho2

1Department of InternalMedicine and Bioengineering,Hanyang University Collegeof Medicine and Engineering,Seoul, Korea2Department of InternalMedicine and Pediatrics,Seoul National UniversityCollege of Medicine,Seoul, Korea3Department of Pediatrics,Yonsei University Collegeof Medicine,Seoul, Korea

∗Correspondence to:Yongsoo Park,Department of InternalMedicine and Bioengineering,Hanyang University Hospital,Seoul 471-020, KoreaE-mail: parkys@hanyang.ac.kr

Accepted: 31 May 2011

AbstractBackground Recently, the association of a common STAT4 haplotype withtype 1 diabetes (T1D) as well as rheumatoid arthritis has been documentedin Caucasians and Koreans. STAT4 is involved in the signalling of interleukin-12 and γ IFN, as well as interleukin-23. To discover genes affecting thesusceptibility of common autoimmune diseases, we studied the associationof polymorphisms in STAT4 with autoimmune thyroid disease (AITD) as wellas T1D in the Korean population.

Subjects and methods Four single-nucleotide polymorphisms on the chro-mosome 2q (rs11889341, rs7574865, rs8179673, and rs10181656), whichwere found to associate with rheumatoid arthritis were examined for associ-ation in a Korean sample of 428 AITD, 418 T1D patients, and 1060 controls.

Results The minor alleles of all four single-nucleotide polymorphisms andthe reconstructed STAT4 haplotypes conferred significant degree of riskfor AITD (p = 10−2 to 10−4). Although we found a weak association ofrs11889341 with T1D (p < 0.05), the same haplotypes were not associatedwith T1D susceptibility. When we stratified T1D patients according to the ageof onset, the minor alleles of all four single-nucleotide polymorphisms andthe same haplotypes showed significant association with the susceptibility ofT1D in the early-onset subgroup (p < 0.01), not in the late-onset subgroup.

Conclusion STAT4 alleles and the same haplotypes might influencecytokine signalling, and therefore the development of AITD as well as T1D.These results reinforce the influence of STAT4 gene as a general autoimmunegene. Copyright 2011 John Wiley & Sons, Ltd.

Keywords STAT4; type 1 diabetes mellitus; autoimmune thyroid diseases;Graves’ disease; Hashimoto’s thyroiditis; rheumatoid arthritis

Introduction

Despite the many unique clinical and serological features associated withspecific autoimmune diseases, the frequent overlap of autoimmune diseasesin individuals or families, coupled with growing evidence that differentautoimmune diseases share susceptibility loci, predict commonalities intheir genetic aetiologies [1]. This prediction is borne out by recent studiesshowing that susceptibilities to type 1 diabetes (T1D), rheumatoid arthritis,and autoimmune thyroid diseases (AITD) are associated with functionalpolymorphisms in general autoimmunity genes, CTLA4 and PTPN22, let alonewell-known human leukocyte antigen (HLA) genes [2,3]. This might alsosuggest that the protein products of these genes are crucial in maintainingcommon immune cellular homeostasis.

Copyright 2011 John Wiley & Sons, Ltd.

868 Y. Park et al.

Graves’ disease and Hashimoto’s thyroiditis share thecharacteristics of AITD which involve infiltration of thethyroid by T and B cells and produce the thyroid autoan-tibodies. Although the exact nature of autoimmunity ofthyroid has not been fully elucidated, there is a wealthof evidence that AITDs develop in genetically susceptibleindividuals, in conjunction with environmental triggers[4]. λs, an indicator of familial aggregation, has beenreported to be between 5.9 and more than 10 in AITDs,suggesting a strong genetic component in the devel-opment of the disease [5]. However, the exact genesinfluencing susceptibility remain to be identified.

T1D is also an autoimmune disease in which immune-mediated destruction of β cells of the pancreas occurs andboth genetic and environmental components play a rolein the development of the disease. The development ofT1D is governed by multiple susceptibility genes with themajor locus in the HLA class II genes, and a number ofminor loci with modest effects [6]. However, the influenceon the genetic risk of all these genes cannot explain totalfamilial aggregation of T1D [7].

Recently, the association of a common STAT4 haplotypewith T1D as well as rheumatoid arthritis has beendocumented in Caucasians and Koreans [8–10]. STAT4is involved in the signalling of interleukin (IL)-12 andγ IFN, as well as IL-23 [11]. The finding of a common riskhaplotype for rheumatoid arthritis and T1D among Asianand Caucasians prompted us to investigate whether thesame haplotype or the gene is involved in the pathogenesisof similar autoimmune disease such as AITD, in whichboth cellular and humoral autoimmunities are importantin aetiology. In this study, we investigated whether fourselected single-nucleotide polymorphisms (SNPs) withinthe STAT4 gene were associated with AITD as well as T1Din the Korean population. We also examined the putativecorrelation between the STAT4 genotype/haplotype andthe clinically defined subset of T1D with and withoutthyroid autoantibodies.

Methods

Study population

A total of 418 Korean patients with T1D and 1060healthy controls were recruited from the Seoul T1DGenetic Consortium [6,10] and a separate 428 AITD (241Graves’ disease and 187 Hashimoto’s thyroiditis) patientswere recruited from Hanyang University Endocrinologyrepository. All the T1D patients (M:F = 182:236)were less than 15 years old and had insulin therapyupon hospital discharge. The mean age at diagnosiswas 7.5 ± 3.9 years with a mean duration of diabetesof 4.3 ± 3.9 years. AITD (M:F = 391:37) was definedas having either Hashimoto’s thyroiditis or Graves’disease. Hashimoto’s thyroiditis and Graves’ disease werediagnosed by endocrinologists clinically and confirmedby abnormal levels of thyroid hormones and positivityfor autoantibodies to thyroid peroxidase, thyroglobulin

and/or TSH receptor, respectively. The study wasapproved by the Institutional Review Board of HanyangUniversity, Seoul, Korea. All patients or their parents andthe controls provided written informed consent.

Autoantibody measurement, HLAtyping, and genotyping

Levels of autoantibody for GAD and IA-2 were quanti-tatively measured by radioligand binding assays usingin vitro-translated antigens as described previously [12].Two hundred and seventy T1D patients (64.6%) and 204T1D patients (48.8%) were positive for GAD and IA-2antibody, respectively. Sixty-nine (16.5%) T1D patientshad autoantibodies to either thyroid peroxidase or thy-roglobulin.

HLA DRB1 and DQB1 were genotyped using PCR-SSOtechniques as described in previous reports [6,12]. AmongT1D patients, 53.9% of patients had two copies of HLA-DRB1-DQB1 haplotypes that were known as susceptiblefactors for T1D [13], 40.8% had 1 copy, and 5.3% had nocopy.

We genotyped four SNPs (rs11889341, rs7574865,rs8179673, and rs10181656) that had already beenreported to be associated with rheumatoid arthritis andT1D [9,10] using TaqMan fluorogenic 5′ nuclease assay.

Statistics

We applied Hardy–Weinberg equilibrium in the controlgroup for each SNP. Association tests for allele/genotypefrequencies of each SNP and reconstructed haplotypesin cases and controls, and linkage disequilibrium (LD)blocks of four SNPs were determined using Haploviewversion 4.1. We also reconstructed haplotypes of fourSNPs using PHASE to determine the individual haplotypethat was analysed for the association with clinical orlaboratory variables such as age at onset, the presence ofautoantibodies, and the combination of susceptible HLAgenotypes. We applied Chi-square test and Wilcoxon’s twosample test for alleles/genotypes or haplotype analysisusing SAS software, version 9.1 (SAS Institute, Cary, NC,USA). p < 0.05 is considered significant.

Results

Association test for each alleleand haplotype of four SNPs with AITDand T1D

Allele and genotype frequencies of the four SNPs were inHardy–Weinberg equilibrium. The four SNPs were tightlylinked to each other, especially three SNPs (rs7574865,rs8179673, and rs10181656) that were in the same LDblock (D′ = 0.99; r2 > 0.93).

Copyright 2011 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2011; 27: 867–871.DOI: 10.1002/dmrr

STAT4 Polymorphism in General Autoimmune Diseases 869

Table 1. Analysis of genotype distribution (Recessive model of risk allele) of four STAT4 SNPs and risk haplotypes(GTC and TCG) in patients with autoimmune thyroid diseases including Graves’ disease and Hashimoto’s thyroiditis,and type 1 diabetes

Autoimmunethyroid Graves’ Hashimoto’s Type 1diseases disease thyroiditis diabetes ControlN(%) N (%) N (%) N (%) N (%)

rs11889341 TT 56 (13.2) 35 (14.6) 21 (11.4) 52 (12.6) 101 (8.8)CC + CT 368 (86.8) 205 (85.4) 163 (88.6) 362 (87.4) 1052 (91.2)OR (95% CI) 1.59

(1.12–2.24)1.78

(1.18–2.69)1.34

(0.82–2.21)1.50

(1.05–2.13)–

p-Value 0.009 0.006 0.25 0.025 –rs7574865 TT 61 (14.3) 33 (13.7) 28 (15.0) 50 (12.0) 106 (10)

GT + GG 367 (85.7) 208 (86.3) 159 (85.1) 368 (88.0) 954 (90)OR (95% CI) 1.5 (1.07–2.1) 1.43

(0.94–2.17)1.58

(1.01–2.48)1.22

(0.86–1.75)–

p-Value 0.019 0.09 0.04 0.27 –rs8179673 CC 61 (14.3) 33 (13.8) 28 (15.1) 57 (13.8) 126 (10.9)

CT + TT 365 (85.7) 207 (86.3) 158 (85.0) 356 (86.2) 1025 (89.1)OR (95% CI) 1.36

(0.98–1.89)1.30

(0.86–1.96)1.44

(0.93–2.24)1.30

(0.93–1.82)–

p-value 0.07 0.21 0.1 0.12 –rs10181615 GG 60 (14.1) 33 (13.7) 27 (14.5) 56 (13.6) 127 (11.1)

CG + CC 367 (85.9) 208 (86.3) 159 (85.5) 357 (86.4) 1025 (88.9)OR (95% CI) 1.32

(0.95–1.83)1.28

(0.85–1.93)1.37

(0.88–2.14)1.27

(0.90–1.77)–

p-Value 0.09 0.24 0.17 0.17 –Haplotype GTC 511 (60.3) 295 (61.5) 216 (58.7) 527 (63.6) 1517 (65.8)

OR (95% CI) 0.78(0.67–0.97)

0.84(0.68–1.02)

0.72(0.58–0.90)

0.92(0.78–1.09)

–

p-Value 0.003 0.08 0.004 0.33 –TCG 322 (37.6) 171 (35.5) 151 (40.4) 289 (34.8) 740 (32.1)OR (95% CI) 1.29

(1.09–1.52)1.17

(0.96–1.44)1.45

(1.16–1.81)1.14

(0.96–1.35)–

p-Value 0.0024 0.13 0.0012 0.12 –

Abbreviations: OR (95% CI), odds ratio (95% confidence interval).

The all minor alleles and genotypes homozygousfor the minor alleles of two SNPs (rs11889341 andrs7574865) and the reconstructed STAT4 haplotypesof three SNPs within the same LD block conferredsignificant degree of risk for AITD (Table 1). TheTCG haplotype conferred significant degree of risk forAITD as a whole [odds ratio = 1.29 (1.09–1.52),p < 0.01], and Hashimoto’s thyroiditis [odds ratio= 1.45 (1.16–1.81), p < 0.01]. The GTC haplotypeshowed protective influence for AITD susceptibility asa whole [odds ratio = 0.78 (0.67–0.97), p < 0.01], andHashimoto’s thyroiditis [odds ratio = 0.72 (0.58–0.90),p < 0.01]. We also found a weak association ofrs11889341 with Graves’ disease [TT versus CC + CT;odds ratio (95% confidence interval) = 1.78 (1.18–2.69),p < 0.01].

In the analysis for the association of four SNPs withthe susceptibility of T1D, we found a weak associationof rs11889341 with T1D [TT versus CC + CT; oddsratio (95% confidence interval) = 1.50 (1.05–2.13),p < 0.05], although other alleles or genotypes (dominantor recessive model, data not shown) among all fourSNPs were not associated with T1D susceptibility. Any ofthe haplotypes did not reveal any significant association

with T1D (Table 1). To address whether these variantsaffected various clinical and laboratory phenotypes ofT1D, we analysed the association of alleles and genotypesin terms of sex, age at diagnosis, family history, thepresence of autoantibodies, and the number of copies ofsusceptible HLA DR-DQ in T1D. We could not observeany significant associations of these alleles/genotypes inthese subgroup analyses (data not shown). However,when we stratified T1D patients into early-onset and late-onset subgroups based on less or more than 7.6 years(median value) of age at diagnosis, the minor alleles andgenotypes homozygous for minor alleles of all four SNPsshowed significant association with the susceptibility ofT1D in the early-onset subgroup, not in the late-onsetsubgroup (Table 2). The haplotype analysis showed thattwo haplotypes (GTC and TCG) conferred comparabledegree of risk for early-onset T1D (Table 2). T1Dpatients with GTC haplotype had decreased risk [oddsratio (95% confidence interval) = 0.73 (0.57–0.94),p < 0.05], while those with TCG had increased risk [oddsratio (95% confidence interval) = 1.43 (1.11–1.85),p < 0.01].

Copyright 2011 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2011; 27: 867–871.DOI: 10.1002/dmrr

870 Y. Park et al.

Table 2. Analysis of frequencies of risk alleles of four single-nucleotide polymorphisms and risk haplotype in type 1diabetes patients with earlier onset less than 7.6 years old compared with controls

Single-nucleotide Risk allele/ Type 1 diabetes Control Odds ratio p-valuepolymorphism/haplotype haplotype onset age <7.6 yrs

rs11889341 T 98 (0.36) 394 (27.6) 1.48 (1.12–1.94) 0.005rs7574865 T 112 (0.41) 432 (32.6) 1.43 (1.09–1.87) 0.008rs8179673 C 112 (0.41) 444 (33.5) 1.37 (1.05–1.79) 0.019rs10181615 G 112 (0.41) 444 (33.5) 1.39 (1.06–1.82) 0.015Haplotype GTC 159 (0.58) 1517 (0.65) 0.73 (0.57–0.94) 0.015

TCG 110 (40.1) 740 (0.32) 1.43 (1.11–1.85) 0.006

Discussion

The development of multiple autoimmune diseases iscontrolled by multiple susceptibility genes, the proteinproducts of which are important in maintaining immunehomeostasis [1–5]. In this report, we present datasupporting the hypothesis that STAT4 polymorphismcorrelates with susceptibility to AITD as well as T1D,mostly in early-onset cases in Korea. The same commonSTAT4 haplotypes confer similar degree of risk forrheumatoid arthritis in both Asian and Caucasianpopulations [8,9]. The finding of a common risk haplotypefor AITD, T1D, and rheumatoid arthritis among Asiansand Caucasians could be a reasonable explanation forthe shared susceptibility. There are some recent studiessuggesting an association of STAT4 polymorphism withautoimmune disease susceptibility in people living inSpain and Greece, in which haplotype information wasnot included [14,15]. There are some more reportssuggesting a shared association of STAT4 in patients withSjogren’s syndrome in Germany and Colombia (personalcommunication).

The STAT4 gene encodes a transcription factor thattransmits signals induced by type 1 IFN, IL-12, and IL-23 [11,16,17]. STAT4 has been a key cytokine drivingautoimmune responses, as demonstrated in STAT4-deficient mice that showed resistance to disease in severalmodels of autoimmunity [16,17]. Given the currenthuman evidences of Th1 and Th17 involvement in chronicinflammation such as in AITD as well as T1D [16,18], itwould be expected that STAT4 may exert its influencethrough a defective signalling in these pathways. In thisstudy, we investigated only four selected SNPs withinthe STAT4 gene. The associated haplotype is locatedprimarily in the third intron of the STAT4 gene, andthe actual functional allele(s) remains to be identified.A full resequencing of the STAT4 gene is in progress,and this will help to direct future studies of splicevariation and/or expression differences that may explainthe disease-associated haplotype [11].

In the joint Genome Wide Association study undertakenrecently to examine a large number of individuals for eachof the seven major autoimmune diseases [19], however,the STAT4 genomic region under investigation was notamong the identified susceptibility loci. Therefore, thetrue influence of the genetic risk of STAT4 as a whole

on total familial aggregation remains to be determined.It might be not so strong and depend on the populationsstudied. Although epidemiologic studies have suggestedthat several genetic variants increase the risk of variousautoimmune diseases, large-scale association studies thatexamine many polymorphisms simultaneously includingHLA in particular, recruiting different ethnic populationstogether, are required to allow better prediction of thegenetic risk. Not only the numbers of samples fromaffected and unaffected subjects, but also the accuratephenotyping and subgrouping of the population arecritical in the success of reliable prediction.

Because our present study investigating the sharedautoimmunity gene hypothesis has demonstrated anassociation of the STAT4 locus with multiple autoimmunediseases and the STAT4 regulates a wide varietyof immune responses, we examined the putativecorrelation between the STAT4 genotype/haplotypes andthe clinically defined subset of T1D with and withoutthyroid autoantibodies, in which no significant associationof any SNP or any haplotype with clinical subgroups wasobtained. It might be caused by our small sample numbersof T1D patients with thyroid autoantibodies (n = 69).Although we could not find significant associations, thereis room for further investigation applying individualclinical phenotypes of AITD in this T1D patients.

Although the available genetic data implicate the com-mon STAT4 variant as a determinant of autoimmunedisease susceptibility, the clinical importance of thesedata remains unclear. The possibility that the STAT4locus contains another disease-associated and possiblycausal variant should be thoroughly examined. In addi-tion, the relevance of this risk variant to additionalautoimmune diseases, autoimmune disease susceptibil-ity in other populations and specific disease subphe-notypes and outcomes needs to be addressed in large,well-characterized cohorts. Such studies will provideopportunities for delineating aetiologically relevant inter-actions between STAT4 and other known autoimmunedisease susceptibility alleles, and for determining whethercombinations of these alleles delineate subclasses ofautoimmune diseases. These findings suggest that ‘com-mon’ autoimmune susceptibility alleles are not sharedamong all autoimmune diseases, but rather among sub-groups of these conditions. This paradigm is speculative

Copyright 2011 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2011; 27: 867–871.DOI: 10.1002/dmrr

STAT4 Polymorphism in General Autoimmune Diseases 871

but is potentially of great relevance to the understandingand clinical management of autoimmune diseases. Fur-ther dissection of the relationship between STAT4 andautoimmune disease susceptibility will provide an excel-lent framework for addressing this genetic polygamy andunravelling the common threads of autoimmune disease.

Conflict of interest

None declared.

References

1. Howson JM, Dunger DB, Nutland S,Stevens H, Wicker LS, Todd JA. A type 1diabetes subgroup with a female bias ischaracterised by failure in tolerance tothyroid peroxidase at an early age and astrong association with the cytotoxic T-lymphocyte-associated antigen-4 gene.Diabetologia 2007; 50: 741–746.

2. Ueda H, Howson JM, Esposito L, et al.Association of the T-cell regulatorygene CTLA4 with susceptibility toautoimmune disease. Nature 2003; 423:506–511.

3. Vang T, Congia M, Macis MD, et al.Autoimmune-associated lymphoid tyro-sine phosphatase is a gain-of-function variant. Nat Genet 2005; 37:1317–1319.

4. Park Y, Kim S, Park S, et al. Differentialexpression of non-islet autoimmunity:comparison of Korean and U.S. patientswith type 1 diabetes. Ann NY Acad Sci2004; 1037: 69–73.

5. Tomer Y, Davies TF. Searching for theautoimmune thyroid disease suscepti-bility genes: from gene mapping togene function. Endocr Rev 2003; 24:694–717.

6. Park Y. Why is type 1 diabetes uncom-mon in Asia? Ann NY Acad Sci 2006;1079: 31–40.

7. She JX, Marron MP. Genetic suscepti-bility factors in type 1 diabetes: linkagedisequilibrium and functional analyses.Curr Opin Immunol 1998; 10: 682–689.

8. Remmers EF, Plenge RM, Lee AT, et al.STAT4 and the risk of rheumatoidarthritis and systemic lupus erythemato-sus. N Engl J Med 2007; 357: 977–986.

9. Lee HS, Remmers EF, Le JM, Kast-ner DL, Bae SC, Gregersen PK. Associ-ation of STAT4 with rheumatoid arthri-tis in the Korean population. Mol Med2007; 13: 455–460.

10. Lee HS, Park HW, Yang SW, Kim DH,Park Y. STAT4 polymorphism is asso-ciated with early-onset type 1 diabetes,but not with late-onset type 1 diabetes.Ann NY Acad Sci 2008; 1150: 93–98.

11. Korman BD, Kastner DL, Gregersen PK,Remmers EF. STAT4: genetics, mecha-nisms, and implications for autoimmu-nity. Curr Allergy Asthma Rep 2008; 8:398–403.

12. Park Y. Functional evaluation of thetype 1 diabetes susceptibility candidategenes. Diabetes Res Clin Pract 2007; 77:S110–S115.

13. Shin HD, Yang SW, Kim DH, Park Y.Independent association of tumornecrosis factor (TNF) polymorphism

with type 1 diabetes susceptibility. AnnNY Acad Sci 2008; 1150: 76–85.

14. Mart ınez A, Varade J, Marquez A, et al.Association of the STAT4 genewith increased susceptibility for someimmune-mediated diseases. ArthritisRheum 2008; 58: 2598–2602.

15. Zervou MI, Mamoulakis D, Panier-akis C, Boumpas DT, Goulielmos GN.STAT4: a risk factor for type 1 diabetes?Hum Immunol 2008; 69: 647–650.

16. Holz A, Bot A, Coon B, Wolfe T,Grusby MJ, von Herrath MG. Disruptionof the STAT4 signaling pathway pro-tects from autoimmune diabetes whileretaining antiviral immune competence.J Immunol 1999; 163: 5374–5382.

17. Miossec P. Interleukin-17 in fashion, atlast: Ten years after its description,its cellular source has been identified.Arthritis Rheum 2007; 56: 2111–2115.

18. Pene J, Chevalier S, Preisser L, et al.Chronically inflamed human tissues areinfiltrated by highly differentiated Th17lymphocytes. J Immunol 2008; 180:7423–7430.

19. The Welcome Trust Case ControlConsortium, Genome-wide associationstudy of 14,000 cases of seven com-mon diseases and 3,000 shared controls.Nature 2007; 447: 661–678.

Copyright 2011 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2011; 27: 867–871.DOI: 10.1002/dmrr