DOI: 10.1111/eci.12463

ORIGINAL ARTICLE Association of IRF8 gene polymorphisms with autoimmune thyroid disease Jiunn-Diann Lin*,†, Yuan-Hung Wang*,‡, Chia-Hung Liu§, Ying-Chin Lin§, Jui-An Lin*, Yuh-Feng Lin*,¶, Kam-Tsun Tang** and Chao-Wen Cheng* * Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, †Division of Endocrinology, Department of Internal Medicine, Shuang-Ho Hospital, School of Medicine, College of Medicine, Taipei Medical University, New Taipei City, Taiwan, ‡Department of Urology, Shuang-Ho Hospital, School of Medicine, College of Medicine, Taipei Medical University, New Taipei City, Taiwan, §Department of Family Medicine, Shuang-Ho Hospital, School of Medicine, College of Medicine, Taipei Medical University, New Taipei City, Taiwan, ¶Division of Nephrology, Department of Internal Medicine, Shuang-Ho Hospital, School of Medicine, College of Medicine, Taipei Medical University, New Taipei City, Taiwan, **Division of Endocrinology and Metabolism, Department of Internal Medicine, Veterans General Hospital, National Yang-Ming University, Taipei, Taiwan

ABSTRACT Background The occurrence of autoimmune thyroid disease (AITD) is known to have a major adverse effect on interferon (INF)-a treatment. The genetic variant of the INF regulatory factor 8 (IRF8), a type 1 INF regulator, is associated with susceptibility to systemic lupus erythematosus and multiple sclerosis. In this study, we investigated possible associations of the IRF8 polymorphisms, rs17445836 and rs2280381, with AITD in an ethnic Chinese population. Material and methods In total, 278 patients with Graves’ disease (GD) and 55 patients with Hashimoto’s thyroiditis (HT), and 252 healthy controls were enrolled. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing were used for genotyping. Results Significantly lower frequencies of the GA genotype and A allele of rs17445836 were found in the HT group than in the control group (P = 0
028, odds ratio (OR) = 4
71 and P = 0
022, OR = 4
40, respectively). Both rs17445836 and rs2280381 were associated with the presence of an antimicrosomal antibody (AmiA), and rs2280381 was also associated with the presence of an antithyroglobulin antibody (ATA) in AITD. Moreover, rs17445836 was associated with the level of AmiA in AITD. Conclusions rs17445836 of IRF8 is a possible genetic variant associated with the development of HT. rs17445836 was associated with the production of thyroid antibody, and the GG genotype of rs17445836 was associated with a higher AmiA titre than the GA genotype. Keywords Autoimmune thyroid disease, Graves’ disease, Hashimoto’s thyroiditis, interferon regulatory factor 8. Eur J Clin Invest 2015; 45: (7): 711–719

Introduction Autoimmune thyroid disease (AITD) is the most common organic-specific autoimmune disease, with a prevalence of about 5% of the general population [1]. Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are two major types of AITDs, both of which are characterized by thyroid lymphocytic infiltration responsive to a thyroid autoantigen, followed by the triggering of diverse immune reactions and different subsequent clinical and pathological features [2]. Despite the various immune mechanisms, it is generally believed that the fundamental pathogenesis of GD and HT can mainly be attributed to an interplay of genetic susceptibility and

environmental exposure, which results in immune intolerance [2,3]. Thus, susceptible genes have been widely studied, and several candidate gene variants were identified, including cytotoxic T-lymphocytic-associated antigen-4, CD40, human leucocyte antigen (HLA), thyroid-stimulating receptor, thyroglobulin, etc. [3–7]. Interferon (INF)-a, a type 1 INF, is widely used to treat chronic hepatitis C [8]. In addition to its antiviral functions by increasing type 1 HLA expression in infected cells and consequent cytotoxic immune reactions, INF-a also activates antibody-mediated immune responses and is strongly associated with the occurrence of AITD [9–11]. Evidence demonstrated that overt AITD occurs in about 5–10%, and subclinical AITD

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takes place in 20–40% of INF-a-treated patients. Thus, INF-a is considered one of the important immune factors inducing AITD [12,13]. INF regulatory factor 8 (IRF8), a member of the IRF family of transcription factors, can interact with other factors to either activate or suppress gene expressions [9]. IRF8 is known to play a crucial role in myeloid cell differentiation, B cell and plasma cell development, germinal centre reactions, antibody formation and regulation of type 1 INF production [14]. Recent genetic association studies showed that rs17445836 of IRF8 was associated with the risk of systemic lupus erythematosus (SLE) and multiple sclerosis (MS) [15]. Other studies also reported that rs2280381 of IRF8 was associated with a susceptibility to SLE [16,17]. However, until now, no studies have been conducted to investigate the relationship between genetic variants of IRF8 and AITD, in spite of the tight correlation between INFa and AITD. Thus, the association of IRF8 with AITD remains unclear. As aforementioned, the correlation between IRF8 and AITD is very interesting, and reports of genetic variants of IRF8 being associated with autoimmune disease are limited. Thus, in this study, we investigated possible associations of single-nucleotide polymorphisms (SNPs) of IRF8, rs17445836 and r2280381, which were shown to be strongly linked to SLE and MS, with AITD in an ethnic Chinese population. At the same time, differences in clinical characteristics between these genetic variants, including disease severity, thyroid function, clinical outcomes and thyroid autoantibody (TAb) titres at the baseline, in GD and HT patients were also determined.

Materials and methods Subjects Blood samples of patients with AITD (278 with GD and 55 with HT) were collected by the Division of Endocrinology and Metabolism of the Internal Department of Wan-Fang (Taipei) and Shuang-Ho Hospitals (New Taipei City, Taiwan) from January 2009 to September 2014. In total, 252 blood samples of subjects without AITD or other autoimmune disease were obtained by the Health Screening Center of Shuang-Ho Hospital from May to August 2014. Control subjects were not sexor age-matched to patients in the GD and HT groups. The study protocol was approved by the hospital’s institutional review board and ethics committee, and all subjects provided written informed consent prior to participation. Graves’ disease was defined by one of the following criteria: (i) the presence of thyrotoxicosis with suppressed thyroidstimulating hormone (TSH), either normal or elevated free thyroxine (FT4), and the presence of a TSH receptor antibody (TSHRAb); (ii) the presence of thyrotoxicosis with a negative TSHRAb but increased or normal diffuse thyroid uptake of

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radioiodine; or (iii) a previous diagnosis by another hospital according to medical records. Intractable GD was defined as TSHRAb remaining positive after treatment with antithyroid drugs (ATDs) for > 24 months and the mediations being unable to be discontinued, while GD in remission was defined as the absence of TSHRAb after treatment with ATDs for ≤ 24 months. At the same time, GD was also divided into two subgroups: GD without the TAb [antimicrosomal antibody (AmiA) and antithyroglobulin antibody (ATA)] and GD with the TAb (either with AmiA or ATA or both). HT was defined as the presence of the TAb (either AmiA or ATA or both) in conjunction with hypo-echogenic thyroid parenchyma detected by a thyroid sonogram. Subjects with HT in a euthyroid status without levothyroxine sodium replacement were regarded as having mild HT, while those in a hypothyroid status with levothyroxine sodium supply were considered to have severe HT. All patients in the three GD, HT and control groups were unrelated. In the GD and HT groups, no patients were found to have either SLE or MS. Reporting of this study conforms to the STROBE statement along with references to the STROBE statement and the broader EQUATOR guidelines [18].

Genotyping Genomic DNA was isolated from 3 mL of EDTA-containing whole-blood samples using a commercial DNA blood kit (Geneaid, XiZhi, New Taipei City, Taiwan). Genotyping was performed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. PCR amplification was performed using 1 lL DNA, 5 lL 109 PCR buffer, 3 lL MgCl2 (1
5 mM), 0
5 lL dNTP (0
2 mM), 1 lL primer and 0
3 lL Taq (1
5 U/50 lL) DNA polymerase (Fermentas, Vilnius, Lithuania). The PCR conditions of the two SNPs were as follows: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s for 30 cycles, with a final 7min extension at 72 °C. The PCR products were incubated with Table 1 Demographic characteristics in Graves’ disease (GD), Hashimoto’s thyroiditis (HT) and control groups Control

GD

HT

AITD

Age (years)

44
1  11
9c

45
0  12
6

48
4  13
6a

45
5  12
8

Sex (female %)

67
5c

70
5

96
4a

74
8

Body mass index (kg/m2)

23
9  3
8

23
3  3
2

22
8  3
7a

Smoking (%)

15
1

20
7

19
6

20
5

29
2a

23
4a

28
0a

Family history of thyroid diseases (%)

6
7b,c,d

b,d

22
7  3
8

a

Age and body mass index are expressed as the mean standard deviation. a p < 0
05 vs. the control group, bp < 0
05 vs. GD, cp < 0
05 vs. HT, dp < 0
05 vs. AITD.

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SNP OF IRF8 IN AITD

Table 2 Genotypes and alleles frequencies for rs17445836 and rs2280381 in the interferon regulatory factor eight gene Polymorphism

Control n (%)

GD n (%)

HT n (%)

AITD n (%)

OR1 (95% CI)

OR2 (95% CI)

OR3 (95% CI)

1

1

1

0
92 (0
58–1
47)

4
71 (4
1–20
1)*

1
08 (0
57–1
45)

1

1

1

rs17445836 38 (15
1)

45 (16
2)

2 (3
6)

47 (14
1)

214 (84
9)

233 (83
8)

53 (96
4)

286 (85
9)

38 (7
5)

45 (8
1)

2 (1
8)

47 (7
1)

466 (92
5)

511 (91
9)

108 (98
2)

619 (92
9)

0
93 (0
59–1
45)

192 (77
1)

206 (74
1)

48 (87
3)

254 (76
3)

1

1

1

52 (20
9)

68 (24
5)

7 (12
7)

75 (22
5)

1
22 (0
21–1
84)

0
54 (0
23–1
26)

1
07 (0
72–1
60)

5 (2
0)

4 (1
4)

0 (0
0)

4 (1
2)

0
73 (0
20–2
82)

57 (22
9)

72 (25
9)

7 (12
7)

79 (23
7)

1
18 (0
79–1
76)

0
49 (0
21–1
15)

1
03 (0
70–1
52)

436 (87
6)

480 (86
3)

103 (93
6)

583 (87
5)

1

1

1

62 (12
4)

76 (13
7)

7 (6
4)

83 (12
5)

1
11 (0
78–1
60)

0
48 (0
21–1
08)

1
03 (0
70–1
52)

GA GG Allele A 4
4 (1
05–18
5)*

1
10 (0
70–1
70)

G rs2280381 TT CT 0
61 (0
16–2
24)

CC CC+CT Allele T C GD, Graves’ disease; HT, Hashimoto’s thyroiditis; AITD, autoimmune thyroid disease (Graves’ disease + Hashimoto’s thyroiditis); control, control group; CI, confidence interval; OR1, odds ratio 1, Graves’ disease vs. the control; OR2, odds ratio 2, Hashimoto’s thyroiditis vs. the control; OR3, odds ratio 3, AITD vs. the control. *P < 0
05.

restriction enzymes (New England Biolabs, Beverly, MA, USA) at 37 °C for 4 h. After incubation, the DNA fragments were detected by electrophoresis with a 3% agarose gel. The primers and restriction enzymes for the two SNP are shown in Table S1. Approximately 10% of the unrelated samples were subjected to repeat genotyping to exclude digestion errors, and no genotyping error was found. In addition, several PCR products were directly sequenced for quality control (Figs S1 and S2).

Laboratory analysis Serum-free T4 and TSH were measured by an electrochemiluminescence immunoassay using commercial Roche Elecsys

reagent kits (Roche Diagnostica, Rotkreuz, Switzerland). The normal range of free T4 was 0
93–1
7 ng/dL (with an lower detection limit of 0
023 ng/dL, an intra-assay coefficient of variation (CV) of < 2
0%, and an interassay CV of < 4
8%), and that of TSH was 0
27–4
20 mU/L (with a sensitivity of 0
014 mU/L, an intra-assay CV of < 3
0%, and an interassay CV of < 7
2%). Serum AmiA and ATA titres were determined by the particle agglutination method using available commercial kits (Fujirebio). A reciprocal titre of ≥ 1 : 100 was considered positive. Serum TSHRAb was measured by a radioimmunoassay method using a commercial TSH receptor autoantibody-coated tube kit (R.S.R.). Results are expressed as the percentage of the

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blocking of I125-labelled TSH binding to the TSH receptor coated on the test tube [6]. A value of > 15% was considered positive.

Statistical analysis Statistical analyses were performed with SPSS vers. 13.0 for Windows (SPSS, Chicago, IL, USA). Quantitative values are shown as the mean (standard deviation; SD). An independent ttest was used to evaluate the demographic data, thyroid function and TSHRAb between two groups. A v2 test or Fisher’s exact test was used to analyse the difference between either the GD or HT group with the control group. A one-way analysis of variance (ANOVA) was used to compare differences in clinical parameters among the GD, HT and control groups. The Bonferroni test was used for post hoc examinations. A test of Hardy– Weinberg equilibrium (HWE) was conducted with the v2 test. The v2 test or Fisher’s exact test was also used to assess differences in categorical data between two groups. As AmiA titres derived from the immunoagglutination method were shown as < 1 : 100, 1 : 100, 1 : 400, 1 : 1600, 1 : 6400, 1 : 25 600, 1 : 102 400 and > 1 : 102 400, it was difficult to compare differences between the two groups. Thus, we arbitrarily divided subjects with AITD into three subgroups according to AmiA levels: low (≤ 1 : 1600), medium (1 : 6400– 1 : 25 600) and high titres (> 1 : 25 600). The association of the susceptibility to HT with clinical variables, including SNPs, and demographic data were first evaluated by a univariate logistic regression. For instance, when HT was defined as the dependent variable (0 for the control, 1 for HT), the variables of interest, that is, SNPs, body mass index (BMI), age, sex, family history (FH) of thyroid diseases and smoking, were taken as the independent variables. Significantly discriminating factors were determined and further put into the multivariate logistic regression for analysis. All statistical tests were two-sided, and a P value of < 0
05 was considered significant.

Results Demographic data of the GD, HT and control groups Patients in the HT group were older than those in the control group, while there was a higher percentage of females than in the GD and control groups. Patients with GD had lower BMI values than those in the control group. There was no difference in the prevalence of smoking among the three groups. Patients in both the GD and HT groups had stronger FH than those in the control group. These results are shown in Table 1.

SNP association analysis Genotype frequencies of rs17445836 and rs2280381 were determined in all groups, and all of them exhibited HWE (GD: rs17445836, P = 0
142; rs2280381, P = 0
544; HT: rs17445836,

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P = 0
891; rs2280381, P = 0
490; AITD: rs17445836, P = 0
167; rs2280381, P = 0
566; and control groups: rs17445836, P = 0
195; rs2280381, P = 0
507). Differences in genotype and allelic frequencies of rs17445836 and rs2280381 in the GD, HT, AITD and control groups were compared, and results are shown in Table 2. There were no significant differences in genotype or allelic frequencies of either rs17445836 or rs2280381 between the GD and control groups or between the AITD and control groups. However, significantly reduced frequencies of the GA genotype and A allele frequencies of rs17445836 were found in the HT group than in the control group (P = 0
028, odds ratio (OR) = 4
71 and P = 0
022, OR = 4
40, respectively). It should be noted that there was a marginally significant difference in C allele frequencies of rs2280381 between the HT and control group (6
4% in the HT group and 12
4% in the control groups, P = 0
068, OR = 0
48). In contrast, there was no significant difference in genotype frequencies between these two groups (P = 0
198).

Associations of rs17445836 and rs2280381 with the presence of the AmiA and ATA in AITD The prevalence of the rs17445836 GG genotype was significantly higher in the AmiA-positive than in the AmiA-negative subgroup (P = 0
032), while there was no difference in the prevalence of the rs17445836 GG genotype between the ATA-positive and ATA-negative groups. However, the percentage of the rs2280381 TT genotype was higher in the AmiA-positive than in the AmiA-negative group (P = 0
013), and it was also higher in the ATA-positive than the ATAnegative group (P = 0
040). The prevalence of the TT genotype in either the ATA-positive or AmiA-positive or bothpositive groups was also higher than those in the absence of both ATA and AmiA (P = 0
035). These results are shown in Table 3.

Prevalence of rs17445836 and rs2280381 in GD without the AmiA and ATA, GD with the AmiA or ATA, and HT Prevalence of rs17445836 and prevalence of rs2280381 in GD without the TAb, GD with the TAb, and HT were also studied, and results are shown in Fig. 1. Percentages of the GA genotype of rs17445836 declined in a gradual way as the disease status changed from GD without the TAb, to GD with the TAb and further to HT (P = 0
036, panel a). There was no significant difference in the prevalence of the GA genotype of rs17445836 between GD with the TAb and GD without the TAb (P = 0
144, data not shown). Similarly, the prevalence of the GA genotype of rs2280301 decreased when the disease status changed from GD without the TAb, to GD with the TAb and finally to the HT group (P = 0
007, panel

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SNP OF IRF8 IN AITD

Table 3 Associations of rs17445836 and rs2280381 with the presence of the antithyroglobulin antibody (ATA) and antimicrosomal antibody (AmiA) in subjects with autoimmune thyroid disease (AITD) AmiA( ) n (%)

AmiA(+) n (%)

P value

ATA ( ) n (%)

ATA(+) n (%)

P value

ATA( )/AmiA ( ) n (%)

ATA(+) or AmiA (+) n (%)

P value

GG

32 (76
2)

213 (88
4)

0
032

26 (86
7)

29 (90
6)

0
703

30 (78
9)

215 (87
8)

0
138

GA

10 (23
8)

28 (11
6)

4 (13
3)

3 (9
4)

8 (21
1)

30 (12
2)

TT

27 (64
3)

196 (81
3)

22 (73
3)

30 (93
8)

25 (65
8)

198 (80
8)

CC+CT

15 (35
7)

45 (18
7)

8 (26
7)

2 (6
3)

13 (34
2)

47 (19
2)

rs17445836

rs2280381 0
013

0
040

0
035

AITD, Graves’ disease + Hashimoto’s thyroiditis. AmiA( ), absence of the antimicrosomal antibody (AmiA); AmiA(+), presence of the AmiA; ATA( ), absence of the antithyroglobulin antibody (ATA); ATA(+), presence of the ATA; ATA( )/AmiA( ), absence of both the ATA and AmiA; ATA(+) or AmiA (+), presence of either the ATA or AmiA or both.

b). The percentage of the GA genotype in GD with the TAb was lower than that without the TAb (P = 0
030, data not shown).

(a)

Associations of rs17445836 and rs2280381 with thyroid function at the baseline and clinical outcomes of GD and HT patients Correlations between the severity of HT at the baseline and the two SNPs were determined. Among 55 patients with HT, one patient who had HT with a thyroid nodule and had previously received a lobectomy was excluded from the analysis. There was no significant association of the severity of HT with the two SNPs. In 142 regularly followed-up patients with GD, no significant difference was shown between remission and the two SNPs. Results are shown in Tables 4 and 5, respectively.

(b)

Associations of rs17445836 and rs2280381 with TSHRAb and AmiA titres There was no significant difference in TSHRAb titres at the baseline between the GA and GG genotypes of rs17445836 or between the CC+CT and TT genotypes (P = 0
169, and P = 0
175, respectively, data not shown). As serum AmiA levels were significantly elevated, the percentage of the GG genotype frequency of rs17445836 also increased (P = 0
032). Meanwhile, there was no association between the serum AmiA titre and the rs2280381 genotype (P = 0
399). The results are shown in Fig. 2.

Multivariate logistic regression analysis for susceptibility to HT Age, sex and FH were shown to have predictive value in the development of HT by the univariate analysis, and the three factors in conjunction with either rs17445836 or rs2280381 were analysed by a multivariate logistic regression. rs17445836 was

Figure 1 Differences in prevalence of rs17445836 (panel a) and prevalence of rs2280381 (panel b) among Graves’ disease (GD) without the thyroid autoantibody (TAb), GD with the TAb, and Hashimoto’s thyroiditis (HT); GD without the TAb, GD without the presence of both the antithyroglobulin antibody (ATA) and antimicrosomal antibody (AmiA); GD with the TAb; and GD with the presence of either the ATA or AmiA.

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Table 4 Associations of thyroid function at the baseline with rs17445836 and rs2280381 in Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) HT

GD

Free T4 (ng/dL)

TSH (lU/mL)

Free T4 (ng/dL)

TSH (lU/mL)

GG

0
94  0
69

18
0  26
9

4
62  1
89

< 0
03

GA

0
72  0
42

31
0  42
5

4
23  1
75

< 0
03

P value

0
658

rs17445836

0
511

0
275

rs2280381 CC+CT

0
75  0
30

21
8  28
1

4
49  1
85

< 0
03

TT

0
96  0
72

18
0  27
4

4
59  1
89

< 0
03

P value

0
466

0
734

0
732

Free T4 and thyroid-stimulating hormone (TSH) values are expressed as the meanstandard deviation.

still retained in the model (P = 0
038, adjusted OR = 8
6, 95% confidence interval (CI) = 1
1–66
7), while rs2280381 was not (P = 0
163, adjusted OR = 0
502, 95% CI = 0
191–1
321, data not shown).

Discussion Recent studies showed that the INF-a pathway may play an important role in the pathogenesis of autoimmune diseases [19,20]. IRF8, a type 1 INFa regulator, was linked to SLE and MS. As aforementioned, genetic variations of rs17445836 and rs22280381 were strongly linked to two autoimmune diseases, SLE and MS. Thus, the two variants might also be linked to

susceptibility to AITD, a kind of autoimmune disease specific to the thyroid. However, genetic associations between the two SNPs of IRF8 and AITD have not previously been reported. In the current study, frequencies of the GA genotype and A allele of rs17445836 in patients with HT were lower than those in healthy controls. These results imply that the rs17445836 genetic variant of IRF8 may be associated with a susceptibility to HT in this ethnic Chinese population. One should note that the AA genotype was totally absent from our study. However, our results are consistent with data from HapMap-HCB and HapMap-JPT studies, both of which demonstrated that the A allele is extremely rare in Asians (0
091% and 0
067%, respectively). As our population size was limited, it is not surprising that the AA genotype was not found in the current study. Interestingly, there was also a borderline significantly lower frequency of the C allele of rs2280381 in the HT group than in healthy controls, which suggests that rs22800381 might also be a susceptibility locus for HT, and further studies with a larger sample size might confirm this observation. In addition to the important role in type 1 IFN signalling, IRF8 is known to essentially control immune system development, both of which can regulate antibody formation [21]. Thus, correlations between an autoantibody and SNPs of IRF8 in autoimmune disease were studied. Chrabot et al. and Li et al. showed that genetic variants of IRF8 were associated with the presence of autoantibodies in patients with SLE [15,17]. As the AmiA and ATA are hallmarks of AITD, the relationship between the SNPs of IRF8 and these TAbs was further analysed. In the current study, we demonstrated that rs17445836 was associated with the appearance of AmiA but not with ATA in patients with AITD. In contrast, rs2280381 genotypes (CC+CT vs. TT) were associated with the occurrence of either the ATA or AmiA. The findings suggest that genetic variations of

Table 5 Associations of rs17445836 and rs2280381 with clinical outcomes in patients with Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) HT

GD

Severe n (%)

Mild n (%)

P value

Intractable n (%)

Remission n (%)

GG

40 (97
6)

12 (92
3)

0
427

87 (84
5)

31 (79
5)

GA

1 (2
4)

1 (7
7)

16 (15
5)

8 (20
5)

38 (92
7)

10 (76
9)

77 (74
8)

28 (71
8)

3 (7
3)

3 (23
1)

26 (25
2)

11 (28
2)

P value

rs17445836 0
480

rs2280381 TT CC+CT

0
200

0
720

Intractable, thyroid-stimulating hormone (TSH) receptor was positive after using antithyroid drugs for > 24 months; Remission, TSH receptor became negative after using antithyroid drugs for ≤ 24 months.

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(a)

(b)

Figure 2 Antimicrosomal antibody (AmiA) titres of rs17445836 (panel a) and rs2280381 (panel b) in autoimmune thyroid disease (AITD). Low titre, AmiA titre of ≤ 1 : 1600. Medium titre, AmiA titre of 1 : 6400–1 : 25 600. High titre, AmiA titre of > 1 : 25 600.

rs17445836 and rs2280381 of IRF8 may be associated with the formation of the AmiA and/or ATA in patients with AITD. There were gradually increasing frequencies of the GG genotype of rs17445836 and the TT genotype of rs2280381 across the spectrum of GD without TAb and GD with TAb to HT, which implies that both variants were associated with AmiA formation in patients with AITD (Fig. 1). Interestingly, there was a significant difference in the genotype frequency of the TT genotype of rs2280381 between the two subgroups of GD, which further suggests that the genetic variant of

rs2280381 in IRF8 might play a crucial role in the formation of the serum AmiA in GD. Differences in TSHRAb levels in GD and AmiA levels in AITD (Fig. 2) at the baseline in rs17445836 and rs2280381 were compared. Our results showed that the rs17445836 GG genotype was associated with higher AmiA levels than the GA genotype, while there was no difference in AmiA levels between the rs2280381 genotypes in patients with AITD. On the contrary, there was no association of TSHRAb levels with the two SNPs in patients with GD. These observations suggest that the rs17445836 genetic variant is associated with the presence of the AmiA and also with AmiA titres in patients with AITD. However, the molecular basis supporting the clinical findings is unclear. Further study to explore the mechanism of TAb formation in AITD is necessary. In the current study, no associations of the two SNPs with clinical characteristics or remission in GD were found (Tables 3 and 4). Despite their association with susceptibility to the development of HT, the two SNPs were not correlated with the clinical severity of HT. As aforementioned, the occurrence of GD and HT is attributed to susceptibility of multiple gene loci and environmental exposure [2,22–24]. Moreover, polygenetic variations in conjunction with environmental factors also contribute to the various phenotypes, clinical severities and responses to treatment [23,24]. Single genetic variations might not be powerful enough to determine clinical features or prognoses of these diseases. Moreover, environmental factors, such as iodine intake, stress, smoking and medications, were not included in the analysis, which also might have affected our results [2,22,24]. To our best knowledge, the current study is the first to demonstrate a possible association of genetic variants of IRF8 with the occurrence of HT, and the presence of the TAb in AITD. However, we should point out certain limitations of the study. First, our sample size was relatively small, and certain demographic information such as FH and smoking were not available for some patients. Further study with a large sample size and complete demographic data could render our results more convincing. Second, both SNPs are located in an intron, which cannot result in changes in amino acids and limits the feasibility of the biological functional assay. Third, evidence showed that IRF-8 modulates IFN-a production, and the intronic SNP might be associated with splicing of RNA and changes in the IRF8 gene messenger RNA level [25], which would subsequently influence the IFN-a concentration and ultimately result in disease development or autoantibody formation [15]. However, serum INF-a levels were not measured in the study, so we were unable to establish correlations among serum INF-a levels and the two SNPs. Finally, we only focused on the two SNPs, rs17445836 and rs2280381, of IRF8 in the present study; it is still possible that other genetic variations of

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IRF8, especially in the adjacent linked coding region, are associated with the development of HT [26]. Thus, it is our future plan to determine associations of other SNPs of IRF8 with AITD. In conclusion, our results show that SNPs of IRF8 are associated with autoimmune thyroid disease. Particularly, rs17445836 of IRF8 may be a susceptible genetic variant for the development of Hashimoto’s thyroiditis. rs17445836 of IRF8 was associated with the presence of the TAb. Moreover, the GG genotype of rs17445836 was associated with higher AmiA titres than the GA genotype. However, only two SNPs were included in our study which cannot completely represent the effect of IRF8. Therefore, additional studies are needed to investigate nearby coding regions of the two SNPs, and INF-a levels should be measured to explore the possible mechanism. Acknowledgements We thank all subjects who participated in the study. This work was supported by a grant from Taipei Medical University and Shuang-Ho Hospital (103TMU-SHH-03) and was partly supported by a grant from Taipei Medical University (TMU100AE3-Y06). The authors thank Mr. Bing-Chun Liu for his technical support. Authors contributions Liu Chia-Hung, Lin Ying-Chin and Lin Jui-An collected data. Lin Jiunn-Diann, Cheng Chao-Wen and Lin Yuh-Feng researched data and contributed to the discussion. Lin JiunnDiann and Cheng Chao-Wen wrote the manuscript. Wang Yuan-Hung and Tang Kam-Tsun reviewed and edited the manuscript. Conflict of interest No conflict of interest. Address Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan (J.-D. Lin, Y.-H. Wang, J.-A. Lin, Y.-F. Lin, C.-W. Cheng); Division of Endocrinology, Department of Internal Medicine, Shuang-Ho Hospital, Taipei Medical University, No.291, Zhongzheng Rd., Zhonghe District, New Taipei City 23561, Taiwan (J.-D. Lin); Department of Urology, Shuang-Ho Hospital, Taipei Medical University, No.291, Zhongzheng Rd., Zhonghe District, New Taipei City 23561, Taiwan (Y.-H. Wang); Department of Family Medicine, Shuang-Ho Hospital, Taipei Medical University, No.291, Zhongzheng Rd., Zhonghe District, New Taipei City 23561, Taiwan (C.-H. Liu, Y.-C. Lin); Division of Nephrology, Department of Internal Medicine, Shuang-Ho Hospital, Taipei Medical University, No.291, Zhongzheng Rd., Zhonghe District,

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New Taipei City 23561, Taiwan (Y.-F. Lin); Division of Endocrinology and Metabolism, Department of Internal Medicine, Veterans General Hospital, No.201, Sec. 2, Shipai Rd., Beitou District, National Yang-Ming University, Taipei 11217, Taiwan (K.-T. Tang). Correspondence to: Chao-Wen Cheng, Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan. Tel.: 886-2-28757514; fax: 886-2-2874-5674; e-mail: [email protected] Received 30 December 2015; accepted 16 May 2015 References 1 Hasham A, Tomer Y. Genetic and epigenetic mechanisms in thyroid autoimmunity. Immunol Res 2012;54:204–13. 2 Tomer Y, Davies TF. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev 2003;24:694–717. 3 Ban Y, Concepcion ES, Villanueva R, Greenberg DA, Davies TF, Tomer Y. Analysis of immune regulatory genes in familial and sporadic Graves’ disease. J Clin Endocrinol Metab 2004;89:4562–8. 4 Ban Y, Davies TF, Greenberg DA, Concepcion ES, Osman R, Oashi T et al. Arginine at position 74 of the HLA-DR beta1 chain is associated with Graves’ disease. Genes Immun 2004;5:203–8. 5 Tomer Y, Concepcion E. Greenberg DA.A C/T single-nucleotide polymorphism in the region of the CD40 gene is associated with Graves’ disease. Thyroid 2002;12:1129–35. 6 Sanders J, Oda Y, Roberts S, Kiddie A, Richards T, Bolton J et al. The interaction of TSH receptor autoantibodies with 125I-labelled TSH receptor. J Clin Endocrinol Metab 1999;84:3797–802. 7 Ban Y, Greenberg DA, Concepcion E, Skrabanek L, Villanueva R, Tomer Y. Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease. Proc Natl Acad Sci USA 2003;100: 15119–24. 8 Mandac JC, Chaudhry S, Sherman KE, Tomer Y. The clinical and physiological spectrum of interferon-alpha induced thyroiditis: toward a new classification. Hepatology 2006;43:661–72. 9 Russo MW, Fried MW. Side effects of therapy for chronic hepatitis C. Gastroenterology 2003;124:1711–9. 10 Parkin J, Cohen B. An overview of the immune system. Lancet 2001;357:1777–89. 11 Burman P, Totterman TH, Oberg K, Karlsson FA. Thyroid autoimmunity in patients on long term therapy with leukocytederived interferon. J Clin Endocrinol Metab 1986;63:1086–90. 12 Tomer Y. Hepatitis C and interferon induced thyroiditis. J Autoimmun 2010;34:J322–6. 13 Tomer Y, Blackard JT, Akeno N. Interferon alpha treatment and thyroid dysfunction. Endocrinol Metab Clin North Am 2007;36:1051– 66 x–xi. 14 Lu R. Interferon regulatory factor 4 and 8 in B-cell development. Trends Immunol 2008;29:487–92. 15 Chrabot BS, Kariuki SN, Zervou MI, Feng X, Arrington J, Jolly M et al. Genetic variation near IRF8 is associated with serologic and cytokine profiles in systemic lupus erythematosus and multiple sclerosis. Genes Immun 2013;14:471–8.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Sequence of the interferon regulatory factor (IRF)-8 single-nucleotide polymorphism (SNP) rs17445836. Figure S2 Sequence of interferon regulatory factor 8 (IRF8) single-nucleotide polymorphism (SNP) rs2280381. Table S1 Single-nucleotide polymorphisms (SNPs) in interferon regulatory factor 8 (IRF8).

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